The overall goal of this method is to trace the contamination of genomic DNA and RNA samples obtained from plant tissue for quantitative real-time PCR. The method uses internal transcribed spacer region of ribosomal genes for reliable and sensitive detection of DNA contamination in Poaceae family. This method can help to improve the widely-applied method of reversed transcription quantitative real-time PCR.
The main advantages are the use of multicopy genes to improve the sensitivity for detection in eukaryotic and prokaryotic species and it's also a cost-effective one. Ribosomal genes consist of the two ITSs namely ITS1 and ITS2, and the three RNA-encoding genes, 17-18S, 5.8S and 25-28S subunits. Here we present a protocol for detection of DNA contamination in RNA samples in the Poaceae species Aeluropus littoralis.
In presented protocol, two complimentary strategies were used for rDNA-based primer design. One, species'specific primers were selected from ITS sequences and two, universal primers were selected from sequences flanking the ITSs. For validation of primer applicability, genomic DNA also should be included in qRT-PCR workflow a crosswidth to RNA isolation and validation.
After isolating DNA and RNA, run a guanidinium thiocyanate agarose gel under denaturing conditions to check the quantity, purity and integrity of the RNA isolated from leaf tissues. To make the gel, first cool the agar to 60 degrees Celsius, and then mix five millimolar guanidinium thiocyanate with standard one-fold TBE 1%agarose gel. Then, prepare the RNA denaturing loading buffer.
Add one to five micrograms of total RNA and RNA denaturing loading buffer. Then, heat the mixture sample at 70 degrees Celsius for five minutes and keep on ice before loading on the gel. Load the sample, and run the gel at 100 volts for 45 minutes.
To see two distinct RNA bands, use an image capture system under ultraviolet light. Remove traces of genomic DNA from RNA in an RNase-free tube by adding one unit of DNASE1 and magnesium chloride containing 10-fold reaction buffer to 0.1 to one microgram of total RNA. Then, incubate the mixture at 37 degrees Celsius for 30 minutes.
Stop the reaction by adding one microliter of 50-millimolar EDTA. And then, incubate the mixture at 65 degrees Celsius for 10 minutes. To remove traces of RNA from genomic DNA extract, add five microliters of 10-milligram-per-milliliters of RNnase A to total DNA and incubate at 37 degrees Celsius for one hour.
Then, store the RNA and DNA extracts at negative 80 degrees Celsius. The functionality of the designed primers should be validated by performing qPCR using gDNA as a template. Prepare the PCR master mix in a tube kept on ice by mixing all of the reagents except the DNA template.
Aliquot the master mix in an optical 96-well plate. Then, add one microliter of genomic DNA to each well, and cover the plate with optical plate sealing film. Spin the plate, and keep in a thermocycler.
Set a program running for 40 cycles, and start the real-time thermocycler. Perform data acquisition at 60 degrees Celsius. To confirm the primer specificity, analyze the melt curve data with single-threshold cycle and subtracted curve fit method.
A sharp individual peak represents a single uniform amplicon. Use a 3%agarose gel to validate the size of each amplicon. Mix five to 10 microliters of PCR product with six-fold loading buffer, and load the samples next to the DNA ladder on the agarose gel.
Conduct electrophoresis at 100 volts for 45 minutes and one-fold tris-boric EDTA buffer. Here is the schematic of the amplification plate and the gDNA contamination assay. Perform all assays in at least three replications.
Check RNase by at least one universal or specific are rDNA-based primer. Include positive control genomic DNA for each primer pair master mix. Include non-template controls for each primer pair master mix.
Prepare the PCR master mix in a tube kept on ice by mixing all the reagents expect the RNA template. Aliquot the master mix in an optical 96-well plate. Then, add one microliter of RNA to each well, and cover the plate with optical plate sealing film.
Centrifuge and keep the plate in the thermocycler. Set a 40-cycle program and start the real-time thermocycler. Analyze the data at 60 degrees Celsius.
Validate all of the amplicons by running a 3%agarose gel. Thaw the RNA treated with DNase and cDNAase synthesis reagents at room temperature. After thawing, spin the reagents.
Then, in a nuclease-free tube, add one microgram of RNA, and one microliter of oligo-deoxythymidine 18 primer, also called Oligo(dT)and adjust the final volume to 12 microliters with RNase-free water. Mix the reagents gently and keep on ice. Incubate the reaction at 65 degrees Celsius for five minute to melt the secondary structures of RNA.
Then, spin the samples and immediately transfer the vial on ice. Add cDNA synthesis reagents to a tube kept on ice. Gently mix the reagents.
Then, add 19 microliters of master mix to a PCR tube containing RNA. Incubate the reaction at 42 degrees Celsius for 60 minutes. And then, at 70 degrees Celsius for five minutes to stop the reserve transcriptase activity.
Then, keep the tubes on ice. Perform routine gene expression analysis of all the PCR reactions by analyzing the melting curves and validating the amplicons on 3%agarose gel. A BLAST search performed to extrapolate the sequence alignment for the small subunit 5.8S and large subunit primers show a motif based on the sequence homology from 2, 000 green plant species.
Representative melt curves done showing single sharp peaks for the internal transcribed spacer-one flanking amplicons in different Poaceae species. The X-axis of the melting curve denotes the temperature and the Y-axis refers to difference in florescence. The peaks in pink color represent the amplicons, and the red line represents the no-template controls in each of the plots.
Representative melt curves showing the presence of sharp peaks with no primer dimers for internal transcribed spacer-two flanking amplicons in different Poaceae species. The X-axis of the melting curve denotes the temperature, and the Y-axis refers to the difference in the florescence. The peaks in green color represent the amplicons, and the orange represent the no-template controls in each of the plots.
A representative 3%agarose gel showing ribosomal DNA-based PCR products running alongside a 100-BP DNA ladder. The DNA bands in panel A represent the amplicon of internal transcribed spacer-one flanking region. DNA bands in panel B and C represent amplicons of internal transcribed spacer-two flanking region.
An internal transcribed spacer-one region for different species. The presence of any bend or peak in RNA samples is the result of gDNA contamination while the appearance of any bend or peak in the NTC reaction is probably related to primer dimer formation. It is recommended to first test all RNA samples by rDNA-based primers to ensure that contaminated samples are not used for downstream applications.
Once mastered, this technique can be completed within eight hours if performed properly. This method has paved the way for researchers to quantify mRNA via quantitative real-time PCR. After watching this video, one should have a good understanding of how genomic DNA contaminations can be detected using our proposed method.
Don't forget that working with guanidinium thiocyanate and ethidium bromide is extremely hazardous and you should always wear protective clothes and take protective measurements.
