We would like to acknowledge Bettine Gibbs for her technical help.

Wear a lab coat, gloves, and safety glasses at all times while performing all procedures in this protocol. Ensure that containers/tubes containing the phenol and guanidine isothiocyanate reagent are only opened in a certified hood, and dispose of phenol waste in a designated container. Use DNAse/RNAse-free sterile tubes, tips, and reagents.
1. Cell Culture
<ol>
	<li>Grow the HeLa cell line and two suspension mantle cell lymphoma cell lines, Granta-519 and Jeko-1, in DMEM containing 10% FBS and 100 U/mL Penicillin/Streptomycin in a humidified incubator with 5% CO2 at 37 &#176;C. Dilute the cells 1:10 and count using a particle counter14.</li>
	<li>For RNA extraction, plate 500,000 cells/well in a 6 well plate (2 mL media per well). After incubation for 24 h, collect RNA from the cells.</li>
</ol>
2. RNA Extraction
<ol>
	<li>Harvesting cells 
	NOTE: With the exception of the centrifugation step, perform all the listed steps in a tissue culture hood to maintain sterility.
	<ol>
		<li>For suspension cells (Jeko-1 and Granta-519):
		<ol>
			<li>Transfer the cells (together with 2 mL of media) to a 15 mL tube, and centrifuge at 1,725 x g for 5 min. Aspirate the media and leave the cell pellet in the bottom of the tube.</li>
			<li>Resuspend the cell pellet in 50 &#181;L of 1x Phosphate Buffered Saline (PBS). Add 500 &#181;L of monophasic phenol and guanidine isothiocyanate reagent to each sample in the 1.5 mL microcentrifuge tube. Mix well and leave at room temperature (RT) for 5 min while inverting the microcentrifuge tube every 1 min.</li>
		</ol>
		</li>
		<li>For adherent cells (HeLa):
		<ol>
			<li>Aspirate the cell culture media from each well. Add 1 mL of PBS to each well to wash off debris.</li>
			<li>Remove the PBS and add 500 &#181;L of monophasic phenol and guanidine isothiocyanate reagent to each well.</li>
			<li>Incubate at RT for 5 min with gentle rocking. Transfer to a 1.5 mL microcentrifuge tube.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Cell lysis
	<ol>
		<li>Freeze the phenol and guanidine isothiocyanate cell mixture at -20 &#176;C for 1 h. 
		NOTE: The mix can be left at -20 &#176;C overnight or until needed.</li>
		<li>Thaw the phenol and guanidine isothiocyanate cell mixture on ice. Add 100 &#181;L of chloroform to the microcentrifuge tube in a PCR hood. Vortex the sample for 10 - 15 s until the mixture is pink and opaque. Incubate the sample on ice (at 4 &#176;C) for 15 min.</li>
		<li>Centrifuge the sample for 15 min at 20,800 x g and 4 &#176;C. Carefully remove the upper colorless aqueous phase (~ 200 &#181;L) and transfer to a new 1.5 mL microcentrifuge tube.</li>
	</ol>
	</li>
	<li>RNA precipitation
	<ol>
		<li>Add an equal volume of isopropanol to each sample. Add 1 &#181;L of coprecipitant (see Table of Materials) to each sample to act as a carrier for the RNA and help visualize the RNA in subsequent steps. Incubate the sample at -80 &#176;C for at least 4 h. For best results, incubate overnight at -80 &#176;C.</li>
		<li>Transfer the sample to a cooled centrifuge. Centrifuge for 35 min at 20,800 x g / 4 &#176;C; the RNA will appear as a blue speck on the bottom of the tube. Carefully remove the isopropanol from the sample. Add 500 &#181;L of 80% ethanol and resuspend the pellet by briefly vortexing for 3 s.</li>
		<li>Centrifuge the sample at 20,800 x g at RT for 5 min. Remove all the ethanol from the sample. Let the sample air-dry for about 10 min. Resuspend the sample in 35 &#181;L of RNAse-free water. Place on a heat block (65 &#176;C) for 5 min to ensure that the RNA is fully in solution, and immediately place the tube on ice.</li>
		<li>Determine the concentration of total RNA using a spectrophotometer as previously described, except use 1.5 &#181;L of the RNA sample instead of 1 &#181;L15. Optionally, run 1 &#181;g of total RNA on a 1% agarose gel to determine the RNA integrity.</li>
	</ol>
	</li>
</ol>
3. DNase Treatment
<ol>
	<li>After quantification of the RNA, perform DNAse treatment on the total RNA to degrade any genomic DNA. For each sample, add the following reagents from the RNAse-free DNAse kit to make a 20 &#181;L total reaction mix in a separate microcentrifuge tube:
	<ol>
		<li>Mix 2 &#181;L of DNase 10X reaction buffer with 4.4 &#181;g of total RNA. Bring to a total of 14 &#181;L with water.</li>
		<li>Add 2 &#181;L of RNase-free DNAse. Incubate at 37 &#176;C for 30 min in the presence of 2 &#181;L of RNase inhibitor (from the Reverse Transcription Kit).</li>
		<li>Add 2 &#181;L of Stop Solution and heat on a heat block for 10 min at 70 &#176;C to inactivate the DNAse enzyme. 
		NOTE: DNAse treatment is optional.</li>
	</ol>
	</li>
</ol>
4. cDNA Synthesis
For a final reaction volume of 50 &#181;L:
<ol>
	<li>For a final reaction volume of 50 &#181;L: Transfer 22 &#181;L of the DNase treated RNA to a new tube, or transfer 4 &#181;g of total RNA with added water to a total volume of 22 &#181;L to a new tube. Add 2 &#181;L of the T7 oligo dT25 primer from 10 &#181;M primer solution. The sequence for the T7 oligo dT25 primer is 5&#39;-GCCGGTAATACGACTCACTATAGTTTTTTTTTTTTTTTTTTTTTTTTT-3&#39;.</li>
	<li>From the Reverse Transcription kit add 2 &#181;L of the RNase inhibitor (20 U/&#181;L), 8 &#181;L of the 5x&#160;Reaction buffer, 4 &#181;L of 10 mM dNTPs, and 2 &#181;L of the Reverse transcriptase (200 U/&#181;L). 
	NOTE: Set up a reaction without reverse transcriptase to act as a negative control.</li>
	<li>Incubate at 42 &#176;C for 1 h. Transfer directly to ice. Heat the tube at 75 &#176;C for 5 min and place the tube on ice.</li>
</ol>
5. Primer Search for Fusion Gene Transcript
<ol>
	<li>Primer design
	<ol>
		<li>Download the cDNA sequence from the Ensemble genome browser (ENST00000360839.6/NM_017747), and identify a region within the ORF of the target transcript (ANKHD1) upstream of the stop codon. Copy and paste the entire region (up to 700 nucleotides) containing sequences upstream of the stop codon into the sequence entry region of the primer design software (&#60;https://www.idtdna.com/Primerquest/Home/Index&#62;).</li>
		<li>Select the show custom design option and select 10 for the number of primers that the system should generate (results to return) and leave all the other parameters set to default.</li>
		<li>Choose five forward primers and two reverse primers that do not overlap. Order primers and reconstitute with RNAse/DNAse-free water to a final concentration of 10 &#181;M. More details about general primer design for PCR are covered elsewhere16.</li>
	</ol>
	</li>
	<li>PCR to identify potential primers for use in subsequent 3&#39; RACE 
	NOTE: To determine the final forward primers to use in the reaction, test the designed primers by setting up different combinations of forward and reverse primers for regular PCR in a PCR hood.
	<ol>
		<li>Transfer the cDNA (2 &#181;L) to a fresh 0.5 mL PCR tube. Add 1 &#181;L of the forward primer and 1 &#181;L of the reverse primer (from a 10 mM primer solution). Make up to 12.5 &#181;L by adding 8.5 &#181;L of nuclease-free water. Add 12.5 &#181;L of the 2x PCR-to-Gel Master Mix.</li>
		<li>Use the following PCR thermal cycling conditions: 95 &#176;C for 2 min followed by 29 cycles of 95 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 1 min. Set the final cycle at 68 &#176;C for 7 min.</li>
		<li>After the PCR, run the products on a 1% agarose gel stained with ethidium bromide. Details of electrophoresis are as previously described with a few modifications17.
		<ol>
			<li>Dissolve 1 g of agarose in 100 mL of Tris-Acetate (TAE) buffer in a 250 mL flask. Boil in a microwave.</li>
			<li>Leave to cool for 1 min and add 7.5 &#181;L of ethidium bromide solution (10 mg/mL stock) in a fume hood. Mix well by swirling the flask and pour into a horizontal gel apparatus. Leave at RT in the fume hood for at least 30 min to allow the gel to solidify.</li>
			<li>Cover the gel with 1x&#160;TAE buffer and load 10 &#181;L of the PCR product onto the agarose gel together with 3 - 5 &#181;L of the DNA molecular weight ladder. Run at 175 V for 10 min. Visualize the products using an imager, and if further separation of products is needed, run the gel for an additional 5 - 10 min.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Selection of nested primers for 3&#39; RACE
	<ol>
		<li>Analyze the results of the PCR agarose gel to select primers that have a distinct, strong, single PCR band. Use the 5&#39;-most primer (ANKHD15&#39;-CCTTCCCAGCGAGTTTCTAC-3&#39;) together with the T7 oligodT25 primer in the first PCR run.</li>
		<li>Select the nested primer that is located downstream of the first primer (ANKHD1 5&#39;-CGTTGGACACAGTGGAATCT-3&#39;), and use it with the T7 primer (5&#39;-GGCCTAATACGACTCACTATAG-3&#39;) in the second set of the PCR reactions.</li>
	</ol>
	</li>
</ol>
6. Optimizing 3&#39; RACE to Map the 3&#39; UTR Using Two Different Enzymes
NOTE: There has been an increase in the diversity of DNA polymerases used for PCR; therefore, we wanted to determine standard conditions that can be applied even when using different enzymes for 3&#39; RACE PCR reactions. The reverse primers for any transcript are kept constant; the only changes are in the nested forward primers that are specific for the target transcript.
<ol>
	<li>Protocol 1: 3&#39; RACE using a modified DNA polymerase from Pyrococcus furiosus (Pfu)
	<ol>
		<li>First PCR
		<ol>
			<li>Transfer 1 &#181;L of cDNA to a PCR tube and add 5 &#181;L of 10x&#160;Pfu reaction buffer. Add 1 &#181;L of the first nested forward primer, 1 &#181;L of the T7 oligodT25 primer, and 1 &#181;L of dNTPs (from a 10 mM solution).</li>
			<li>Make up to 49 &#181;L total volume by adding 40 &#181;L of water. Add 1 &#181;L of Pfu DNA polymerase and mix well. Run the PCR using the PCR profile in Table 1.</li>
		</ol>
		</li>
		<li>Second PCR
		<ol>
			<li>Transfer 2 &#181;L of the products from the first PCR to a new PCR tube and mix with 5 &#181;L of 10x&#160;PfuUltra II reaction buffer. Add 1 &#181;L of the second nested PCR primer, 1 &#181;L of the T7 primer, and 1 &#181;L of dNTPs (10 mM solution).</li>
			<li>Make up to 49 &#181;L total reaction volume by adding 39 &#181;L of water. Add 1 &#181;L of Pfu DNA polymerase. Run the PCR using the same PCR profile as the first PCR setup (Table 1).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Protocol 2: 3&#39; RACE using a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase PCR master mix
	<ol>
		<li>First PCR
		<ol>
			<li>Transfer 1 &#181;L of cDNA to a PCR tube. Add 1 &#181;L of the first nested forward primer and 1 &#181;L of the T7 oligodT25 primer. Make up to 25 &#181;L by adding 22 &#181;L of water.</li>
			<li>Add 25 &#181;L of 2x&#160;PCR Master Mix and mix well. Use the PCR cycling conditions described in Table 2.</li>
		</ol>
		</li>
		<li>Second PCR
		<ol>
			<li>Transfer 2 &#181;L of the products from the first PCR to a new PCR tube. Add 1 &#181;L of the second nested PCR primer together with 1 &#181;L of the T7 reverse primer.</li>
			<li>Make up to 25 &#181;L by adding 22 &#181;L of water. Add 25 &#181;L of 2X PCR Master Mix. Run the PCR using the same PCR profile as the first PCR setup (Table 2).</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
7. Verify the second PCR product of 3&#39; RACE.
<ol>
	<li>Take 5 - 10 &#181;L of the second PCR product (as well as product from the first round of PCRs if the transcript is abundantly expressed) and run on an agarose gel. Set up the gel and run the electrophoresis as previously described (steps 5.2.3.1 to 5.2.3.3). Visualize results on an imager.</li>
</ol>
8. Product Purification and Sequencing
<ol>
	<li>Purify the gel PCR products from the second PCR using the Gel and PCR Fragment DNA Clean-Up System as per the manufacturer&#39;s protocol.</li>
	<li>Perform Sanger sequencing (alternatively, send the gel purified PCR product samples and appropriate sequencing primers to a sequencing lab). Analyze the sequencing data after Sanger sequencing.</li>
	<li>Download sequence-analyzing software (see Table of Materials) and import the trace files into the software. Use the individual PURE Base QVs (Quality Values) to obtain the Base call information18. Download the chromatogram with high quality traces for viewing. 
	Optional: The gel purified product can be cloned using an appropriate cloning kit and the isolated clones sent for Sanger sequencing.</li>
</ol>

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The authors have nothing to disclose.

Despite the advent of massive parallel sequencing technologies, on a gene-by-gene basis, 3&#39; RACE still remains the easiest and most economical method to identify the PAS and nucleotides adjacent to the poly(A) tail. The adaptation described here expands using 3&#39; RACE to both amplify and map sequences that include a portion of the ORF, the stop codon, and the entire 3&#39; UTR of the ANKHD1 mRNA transcript. A major advantage of 3&#39; RACE is that with a few minor adaptations, products from 3&#39; RACE ca...

The formation of the 3&#39; end is a critical step in mRNA maturation that comprises the cleavage of the pre-mRNA downstream of a PAS followed by the addition of ~250 untemplated adenines, which make up the poly(A) tail1,2. The poly(A) binding protein (PABP) binds to the poly(A) tail, and this protects the mRNA transcript from degradation, and facilitates translation1.
Current estimates suggest that 70% of human genes have multiple PASs, and thus undergo alternative polyadenylation, resulting in multiple 3&#39; ends3. Thus, it is important to identify where the poly(A) tail attaches to the rest of the 3&#39; UTR, as well as identify the PAS used by any given transcript. The advent of next-generation sequencing has resulted in the simultaneous identification of the 3&#39; UTRs and the PASs of thousands of genes. This increase in sequencing capability has required the development of bioinformatic algorithms to analyze data involving alternative polyadenylation of the 3&#39; end. For the de novo detection or validation of the PAS and hence mapping of the 3&#39; end of individual genes from large scale sequencing data, 3&#39; RACE remains the method of choice4,5. The sequences included in cDNA products of 3&#39; RACE normally include only a portion of the 3&#39; UTR that contains the poly(A) tail, the cleavage site, the PAS, and the sequences upstream of the PAS. Unlike PCR, which requires the design and use of gene specific forward and reverse primers, 3&#39; RACE only requires two gene specific nested forward primers. Hence, PCR requires a more detailed knowledge of the nucleotide sequence of a large region of the gene being amplified4,6. Since 3&#39; RACE uses the same reverse primer that targets the poly(A) tail for all polyadenylated RNA transcripts, only the forward primers need to be gene specific, thus, only requiring knowledge of a significantly smaller region of the mRNA. This enables the amplification of regions whose sequences are not fully characterized4,7. This has allowed 3&#39; RACE to be used not only to determine the 3&#39; end of a gene, but to also determine and characterize large regions upstream of the PAS that form a significant portion of the 3&#39; UTR. By combining 5&#39; RACE with the modified 3&#39; RACE that includes larger portions of the 3&#39; UTR and flanking regions, it is possible to fully sequence or clone an entire mRNA transcript from the 5&#39; end to its 3&#39; end8.
An example of this application of modified 3&#39; RACE is the recent identification of a novel CCND1-MRCK fusion gene transcript from Mantle Cell Lymphoma cell lines and cancer patients. The 3&#39; UTR consisted of sequences from both the CCND1 and MRCK genes and was recalcitrant to miRNA regulation9. The two nested CCND1 specific forward primers were complementary to the region immediately adjacent and downstream of the CCND1 stop codon. Although whole transcriptome sequencing together with specific bioinformatic tools can be used to detect gene fusions within the 3&#39; UTR10, many labs may lack the financial resources or bioinformatic expertise to make use of this technology. Hence, 3&#39; RACE is an alternative for de novo identification and validation of novel fusion genes involving the 3&#39; UTR. Considering the drastic increase in the number of reported fusion genes as well as read through transcripts, 3&#39; RACE has become a powerful tool in characterizing gene sequences11,12. In addition, recent studies have shown that different sequences within the 3&#39; UTR as well as the length of the 3&#39; UTR can affect mRNA transcript stability, localization, translatability, and function13. Due in part to an increased interest in mapping the transcriptome, there has been an increase in the number of different DNA polymerases being developed for use in the lab. It is important to determine what types of modifications can be made to the 3&#39; RACE protocol in order to utilize the available repertoire of DNA polymerases.
This work reports adapting 3&#39; RACE to map the entire 3&#39; UTR, the PAS, and the 3&#39; end cleavage site of the ANKHD1 transcript by using nested primers within the ANKHD1 section of the transcript and&#160;two different DNA polymerases.

<table>
	<tbody>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>Cervical cancer cell line.</td>
		</tr>
		<tr>
			<td>Jeko-1 cells</td>
			<td>ATCC</td>
			<td>CRL-3006</td>
			<td>Mantle cell lymphoma cell line.</td>
		</tr>
		<tr>
			<td>Granta-519 cells</td>
			<td>DSMZ</td>
			<td>ACC-342</td>
			<td>Mantle cell lymphoma cell line.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Fetal bovine serum for cell culture.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisherScientific</td>
			<td>15140122</td>
			<td>Antibiotic and antimycotic.</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9545</td>
			<td>Coprecipitant.</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisherScientific</td>
			<td>10569044</td>
			<td>Gibco brand cell culture media with GlutaMAX.</td>
		</tr>
		<tr>
			<td>Nuclease Free-Water</td>
			<td>ThermoFisherScientific</td>
			<td>AM9938</td>
			<td>Ambion DNase and RNAse free water(non DEPC treated).</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Solution</td>
			<td>Corning</td>
			<td>21-030</td>
			<td>1X PBS.</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma&#160; Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Sigma Aldrich</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Alcohol</td>
			<td>Sigma Aldrich</td>
			<td>793175</td>
			<td>Ethanol</td>
		</tr>
		<tr>
			<td>Ethidium Bromide solution</td>
			<td>Sigma Aldrich</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisherScientific</td>
			<td>15596026</td>
			<td>Monophasic phenol and guanidine isothiocyanate reagent.</td>
		</tr>
		<tr>
			<td>2X Extender PCR-to-Gel Master Mix</td>
			<td>Amresco</td>
			<td>N867</td>
			<td>2X PCR-to-Gel Master Mix&#160; containing loading dye used in routine quick PCR assays and primer search.</td>
		</tr>
		<tr>
			<td>10mM dNTP</td>
			<td>Amresco</td>
			<td>N557</td>
			<td></td>
		</tr>
		<tr>
			<td>RQ1 RNase-Free DNase</td>
			<td>Promega</td>
			<td>M6101</td>
			<td>Dnase treatment kit.</td>
		</tr>
		<tr>
			<td>Gel Loading Dye Orange (6X)</td>
			<td>New England BioLabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Phusion High-Fidelity PCR Master Mix with HF buffer</td>
			<td>ThermoFisherScientific</td>
			<td>F531S</td>
			<td>2X PCR MasterMix containing a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase and other reagents.</td>
		</tr>
		<tr>
			<td>PfuUltra II Fusion HS DNA polymerase</td>
			<td>Agilent Technologies</td>
			<td>600670</td>
			<td>Modified DNA Polymerase from Pyrococcus furiosus (Pfu).</td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo Fischer</td>
			<td>K1691</td>
			<td>Used for&#160; reverse transcription of mRNA into&#160; cDNA synthesis. Kit includes&#160; Ribolock RNAse inhibitor, RevertAid M-MuLV reverse transcriptase and other reagents listed in manuscript.</td>
		</tr>
		<tr>
			<td>Pefect size 1Kb ladder</td>
			<td>5 Prime</td>
			<td>2500360</td>
			<td>Molecular weight DNA ladder.</td>
		</tr>
		<tr>
			<td>Alpha Innotech FluorChem Q MultiImage III</td>
			<td>Alpha Innotech</td>
			<td></td>
			<td>Used to visualise ethidium bromide stained agarose gel.</td>
		</tr>
		<tr>
			<td>Low Molecular Weight Ladder</td>
			<td>New England BioLabs</td>
			<td>N3233L</td>
			<td>Molecular weight DNA ladder.</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>MidSci</td>
			<td>VM-3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>MidSci</td>
			<td>C1008-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Bath</td>
			<td>MidSci</td>
			<td>DB-D1</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000C</td>
			<td>ThermoFisherScientific</td>
			<td>ND-2000C</td>
			<td>Spectrophotometer.</td>
		</tr>
		<tr>
			<td>Wide Mini-Sub&#160;Cell GT Horizontal Electrophoresis System</td>
			<td>BioRad</td>
			<td>1704469</td>
			<td>Electrophoresis equipment-apparatus to set up gel</td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BioRad</td>
			<td>1645050</td>
			<td>Power supply for gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Dot Scientific</td>
			<td>AGLE-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler Gradient</td>
			<td>Eppendorf</td>
			<td>950000015</td>
			<td>PCR thermocycler.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Fragment DNA Clean-Up System</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Zero Blunt TOPO PCR Cloning Kit, with One Shot&#160; TOP10 Chemically Competent E. coli cells</td>
			<td>ThermoFisherScientific</td>
			<td>K280020</td>
			<td></td>
		</tr>
		<tr>
			<td>MyPCR Preparation Station Mystaire</td>
			<td>MidSci</td>
			<td>MY-PCR24</td>
			<td>Hood dedicated to PCR work.</td>
		</tr>
		<tr>
			<td>Hamilton SafeAire II fume hood</td>
			<td>ThermoFisherScientific</td>
			<td></td>
			<td>Fume hood.</td>
		</tr>
		<tr>
			<td>Beckman Coulter Z1&#160;Particle&#160;Counter</td>
			<td>Beckman Coulter</td>
			<td>6605698</td>
			<td>Particle counter. For counting cells before plating&#160; for RNA extraction.</td>
		</tr>
		<tr>
			<td>Applied Biosystems Sequence Scanner Software v2.0</td>
			<td>Applied Biosystems (through ThermoFisherScientific)</td>
			<td></td>
			<td>Software to analyze Sanger sequencing data.</td>
		</tr>
	</tbody>
</table>

adapting 3' rapid amplification of cdna ends to map transcripts in cancer

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the traditional method of choice is 3&#39; rapid amplification of cDNA ends (3&#39; RACE). Protocols for 3&#39; RACE require the careful design and selection of nested primers within the 3&#39; untranslated region (3&#39; UTR) of the target gene of interest. However, with a few modifications the protocol can be used to include the entire 3&#39; UTR and sequences within the open reading frame (ORF), providing a more comprehensive picture of the relationship between the ORF and the 3&#39; UTR. This is in addition to identification of the polyadenylation signal (PAS), as well as the cleavage and polyadenylation site provided by conventional 3&#39; RACE. Expanded 3&#39; RACE can detect unusual 3&#39; UTRs, including gene fusions within the 3&#39; UTR, and the sequence information can be used to predict potential miRNA binding sites as well as AU rich destabilizing elements that may affect the stability of the transcript.

Nested Forward Primer Search:
The agarose gel from Figure 1 shows two distinct PCR gel products (Lanes 1 and 2) which use the same forward primer but different reverse primers. Lane 3 has a distinct PCR product and has a distinct forward and reverse primer. The ideal primers to use for the PCR based reaction are those that give one distinct PCR product (Lane 3). The forward primer used...

Watch this Scientific Journal Video about Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer at JoVE.com

Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

The two different 3&#39; rapid amplification of cDNA ends (3&#39; RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3&#39; UTR of a transcript using RNA obtained from different cancer cell lines.

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

adapting-3-rapid-amplification-cdna-ends-to-map-transcripts

Research

JoVE Journal

Genetics

12.2K Views.  Butler University. The two different 3' rapid amplification of cDNA ends (3' RACE) protocols described here make use of two different DNA polymerases to map sequences that include a segment of the open reading frame (ORF), the stop codon, and the entire 3' UTR of a transcript using RNA obtained from different cancer cell lines.

Video: Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 nucleotides apart result in either the inclusion (S) or exclusion (&#916;S) of a single residue, Serine-701. Further downstream, splicing at a pair of alternative acceptor splice sites result in transcripts encoding either the 55 terminal residues of the transactivation domain (&#945;) or a truncated transactivation domain with 7 unique residues (&#946;). As outlined in this manuscript, measuring the proportions of STAT3's four spliced transcripts (S&#945;, S&#946;, &#916;S&#945; and &#916;S&#946;) was possible using absolute qPCR (quantitative polymerase chain reaction). The protocol therefore distinguishes and measures highly similar splice variants. Absolute qPCR makes use of calibrator plasmids and thus specificity of detection is not compromised for the sake of efficiency. The protocol necessitates primer validation and optimization of cycling parameters. A combination of absolute qPCR and efficiency-dependent relative qPCR of total STAT3 transcripts allowed a description of the fluctuations of STAT3 splice variants' levels in eosinophils treated with cytokines. The protocol also provided evidence of a co-splicing interdependence between the two STAT3 splicing events. The strategy based on a combination of the two qPCR techniques should be readily adaptable to investigation of co-splicing at other tandem splicing sites.

Tandem splicing events occur at sites less than 12 nucleotides apart. Quantifying ratios of such splice variants is feasible using an absolute quantitative PCR approach. This manuscript describes how splice variants of the gene STAT3, in which two splicing events results in Serine-701 inclusion/exclusion and &#945;/&#946; C-termini, can be quantified.

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription elongation (TE)&#8212;we call such cancers as TEdeff. Notably, TEdeff cancers are characterized by spurious transcription and faulty mRNA processing in a large set of genes, such as interferon/JAK/STAT and TNF/NF-&#954;B pathways, leading to their suppression. The TEdeff subtype of tumors in renal cell carcinoma and metastatic melanoma patients significantly correlate with poor response and outcome in immunotherapy. Given the importance of investigating TEdeff cancers&#8212;as it portends a significant roadblock against immunotherapy&#8212;the goal of this protocol is to establish an in vitro TEdeff mouse model to study these widespread, non-genetic transcriptional abnormalities in cancers and gain new insights, novel uses for existing drugs, or find new strategies against such cancers. We detail the use of chronic flavopiridol mediated CDK9 inhibition to abrogate phosphorylation of serine 2 residue on the C-terminal repeat domain (CTD) of RNA polymerase II (RNA Pol II), suppressing the release of RNA Pol II into productive transcription elongation. Given that TEdeff cancers are not classified under any specific somatic mutation, a pharmacological model is advantageous, and best mimics the widespread transcriptional and epigenetic defects observed in them. The use of an optimized sublethal dose of flavopiridol is the only efficacious strategy in creating a generalizable model of non-genetic widespread disruption in transcription elongation and mRNA processing defects, closely mimicking the clinically observed TEdeff characteristics. Therefore, this model of TEdeff can be leveraged to dissect, cell-autonomous factors enabling them in resisting immune-mediated cell attack.

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

The protocol details an in vitro murine carcinoma model of non-genetic defective transcription elongation. Here, chronic inhibition of CDK9 is used to repress productive elongation of RNA Pol II along pro-inflammatory response genes to mimic and study the clinically observed TEdeff phenomenon, present in about 20% of all cancer types.

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the others contain 5&#39; monophosphate generated post-transcriptionally (processed transcripts). To discriminate between the two types of transcripts, several strategies have been developed, and most of them depend on presence/absence of 5&#39; triphosphate. However, the triphosphate at primary 5&#39; termini is unstable, and it hinders a clear discrimination of the two types of transcripts. To systematically differentiate and map the primary and processed transcripts stably accumulated in maize mitochondrion, we have developed a circular RT-PCR (cRT-PCR)-based strategy by combining cRT-PCR, RNA 5&#39; polyphoshpatase treatment, quantitative RT-PCR (RT-qPCR), and Northern blot. As an improvement, this strategy includes an RNA normalization step to minimize the influence of unstable 5&#39; triphosphate.
In this protocol, the enriched mitochondrial RNA is pre-treated by RNA 5&#39; polyphosphatase, which converts 5&#39; triphsophate to monophosphate. After circularization and reverse transcription, the two cDNAs derived from 5&#39; polyphosphatase-treated and non-treated RNAs are normalized by maize 26S mature rRNA, which has a processed 5&#39; end and is insensitive to 5&#39; polyphosphatase. After normalization, the primary and processed transcripts are discriminated by comparing cRT-PCR and RT-qPCR products obtained from the treated and non-treated RNAs. The transcript termini are determined by cloning and sequencing of the cRT-PCR products, and then verified by Northern blot.
By using this strategy, most steady-state transcripts in maize mitochondrion have been determined. Due to the complicated transcript pattern of some mitochondrial genes, a few steady-state transcripts were not differentiated and/or mapped, though they were detected in a Northern blot. We are not sure whether this strategy is suitable to discriminate and map the steady-state transcripts in other plant mitochondria or in plastids.

We present a circular RT-PCR-based strategy by combining circular RT-PCR, quantitative RT-PCR, RNA 5&#39; polyphosphatase-treatment, and Northern blot. This protocol includes a normalization step to minimize the influence of unstable 5&#39; triphosphate, and it is suitable for discriminating and mapping the primary and processed transcripts stably accumulated in maize mitochondrion.

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding sites for transcriptional regulators to modulate gene expression. Alterations of CREs have been implicated in various diseases including cancer. While promoters and enhancers have been the primary CREs for studying gene regulation, very little is known about the role of silencer, which is another type of CRE that mediates gene repression. Originally identified as an adaptive immunity system in prokaryotes, CRISPR/Cas9 has been exploited to be a powerful tool for eukaryotic genome editing. Here, we present the use of this technique to delete an intronic silencer in the human RUNX1 gene and investigate the impacts on gene expression in OCI-AML3 leukemic cells. Our approach relies on electroporation-mediated delivery of two preassembled Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complexes to create two double-strand breaks (DSBs) that flank the silencer. Deletions can be readily screened by fragment analysis. Expression analyses of different mRNAs transcribed from alternative promoters help evaluate promoter-dependent effects. This strategy can be used to study other CREs and is particularly suitable for hematopoietic cells, which are often difficult to transfect with plasmid-based methods. The use of a plasmid- and virus-free strategy allows simple and fast assessments of gene regulatory functions.

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Direct delivery of preassembled Cas9/guide RNA ribonucleoprotein complexes is a fast and efficient means for genome editing in hematopoietic cells. Here, we utilize this approach to delete a RUNX1 intronic silencer and examine the transcriptional responses in OCI-AML3 leukemic cells.

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). In HIV-1, the antisense protein ASP gene is located on the negative strand of env, in the reading frame -2, spanning the junction gp120/gp41. In the sense orientation, the 3&#39; end of the ASP open reading frame overlaps with gp120 hypervariable regions V4 and V5. The study of ASP RNA has been thwarted by a phenomenon known as RT-self-priming, whereby RNA secondary structures have the ability to prime RT in absence of the specific primer, generating non-specific cDNAs. The combined use of high RNA denaturation with biotinylated reverse primers in the RT reaction, together with affinity purification of the cDNA onto streptavidin-coated magnetic beads, has allowed us to selectively amplify ASP RNA in CD4+ T cells derived from individuals infected with HIV-1. Our method is relatively low-cost, simple to perform, highly reliable, and easily reproducible. In this respect, it represents a powerful tool for the study of antisense transcription not only in HIV-1 but also in other biological systems.

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

RNA hairpins and loops can function as primers for reverse transcription (RT) in absence of sequence-specific primers, interfering with the study of overlapping antisense transcripts. We have developed a technique able to identify strand-specific RNA, and we have used it to study HIV-1 antisense protein ASP.

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...