With the use of this protocol researchers can identify novel exome or extracellular vesicle based and tissue based micro cellular for advanced prostate cancer. Isolating RNA from both F-F-B tissues and extracellular vesicles is often challenging as most of it is either de created or is in limited quantity This particle will elaborate on different methods to optimize the R-N-A inputs and C-D-N-A libraries to gene most specific free and high quality data for next generation sequencing. Next gen sequencing offers the most comprehensive platform for understanding molecule alteration in tumor that underlie tumor progression this particle is optimized with prostate cancer clinical sample, and can provide novel insights into the underlying biology of advanced aggressive prostate cancer.
Optimizing the C-D-N-A libraries from low input material is a crucial step for a successful sequencing run it is therefore very important to generate high quality C-D-N-A libraries to get accurate results. After performing micro dissection of tissues as previously done proceed to isolating serum derived E-V's Thaw rows of patient serum samples at four degrees celsius. Transfer 110 micro liters of thawed serum sample to a tube and spin at two thousand times G for thirty minutes.
Elect the super naden for subsequent E-V or exosome isolation and discard the debree pellet. At 30 microleters of serum exosome isolation reagent to the super natent. An incubate it four degree celsius for thirty minutes.
Spin at ten thousand times G for ten minutes. Elect the E-V super nadent carefully without disturbing the pellet in a separate 1.5 milliliter tube and store minus 80 degree Celsius for future analysis. Then re suspend the E-V pellet in 70 micro liters of P-B-S prepared in nuclease free water.
After isolating total R-N-A from micro dissected tissues, and purified E-V's using commercial kits perform Library Preparation into five micro liters of a normalized forty nanogram per micro liter R-N-A sample at one micro liter of three prime adapter. Pre-heat the thermal cycler to 70 degree celsius and incubate the sample for two minutes. Immediately transfer the samples on ice.
In a separate tube mix two micro liters of ligation buffer, one micro liter of RNase inhibitor and one micro liter of T4 RNA ligase two, a deletion mutant. Mix bye pie bedding up and down. Then add four micro liters of the mix to the tube with the R-N-A 3 prime adapter on ice.
Preheat the thermal cycler to 28 degree Celsius. And place the tube in the thermal cycler to incubate for one hour. After that, add one micro liter of stop solution into the tube while keeping the sample on the thermal cycler.
Incubate at 28 degrees Celsius for another 15 minutes. Then immediately remove the tube and keep on ice. In a separate tube add one microliter of five prime adapter.
Transfer to a thermal cycler that's been preheated to 70 degrees Celsius. And incubate for two minutes. Immediately place the tube on ice to prevent reannealing of the adapters.
Add one micro liter of ten millimole A-T-P to the tube of D natured five prime adapter. Mix bye pie pedding and add one micro liter of T4 R-N-A ligase to the mix. Mix bye pie pedding.
Transfer three microliters of this mix to the tube containing the three prime adapter ligated R-N-A. Place the tube on the thermal cycler that has been pre heated to 28 degrees celsius and incubate for one hour. After that immediately transfer the tube onto ice and either proceed further with the samples or store at minus 80 degrees Celsius for future use.
To perform R-T-P-C-R, use six micro ligers of each adapter ligated R-N-A and add one micro liger of R-N-A R-T primer to it. Transfer the tube to the thermal cycler preheated to 70 degrees Celsius and incubate for two minutes. Transfer the tube to ice.
In a separate tube mix two micro liters of 5X, strand buffer, 5 micro liters of 12.5 milimoler DNTP's, one micro liter of 100 milimolar DTT, one micro liter of R-N-A's inhibitor, and one micro liter of reverse transcriptase. Add five microliters of this mix to the adapter ligated RNA and transfer it to the thermal cycler that has been preheated to 50 degrees Celsius to incubate for one hour. After one hour transfer the adapter ligated complimentary DNA immediately to ice.
In a separate tube, mix 25 microliters of PCR mix, two micro liters of RNA PCR primer, two micro liters of RNA PCR primer index, and 8.5 microliters of RNA's free water. Add 37.5 microliters of this mix to 12.5 microliters of the adapter ligated CDNA. Transfer the tube to the thermal cycler that has been preheated to 98 degrees celsius.
Program the thermal cycler as suggested in the manufacturer's protocol with the cycle number modified to 15. After completion, keep the sample at four degree celsius. Proceed or store at minus 80 degree celsius for future use.
To purify the ligated complimentary DNA, add ten microliters of the DNA loading dye to each complimentary DNA sample. And to ten microliters of the high resolution latter and custom RNA latter. Run 30 microliters of each sample in two separate lanes adjacent to each other in the six percent TBE Polyacrylamide gel.
With custom RNA latter and high resolution latter, the intermediate space between the two different samples. Run the gel in one X TBE buffer at 145 volts for approximately 30 to 40 minutes. Keep track of the lower blue front of the loading dye.
Stop the electrophoresis when it approaches the bottom of the gel. Transfer the gel in one X TBE buffer with 5 microgram per millimeter ethidium bromide. Visualize the gel in a transilluminator and cut the gel precisely between the 145 base pair and 160 base pair markers and slightly above the 145 base pair marker to avoid contamination with adapter dimer.
A critical step is purification ligated CDNA that is done by cutting the gel precisely as adapter dimers run very lose to the desired brand. Transfer the gel pieces to DNA breaking tubes kept in two millimeter collecting tubes. Spin at 20 thousand times G for two minutes.
And add three hundred micro liters of RNase-free water and allow it to rotate gently on a rocker overnight at room temperature. To perform the DNA precipitation on the following day transfer the contents of the tube to 5 micron filter tubes. Spin 600 times G for strictly ten seconds.
Then add two microliters of glycogen thirty microliters of three molar sodium acetate, 1X pellet paint, and 975 microliters of 100 percent ethanol to eluted DNA. Spin at 17, 000 times G at 4 degrees Celsius for 20 minutes. Discard the supernatant and add 75 percent ethanol to wash the pellet.
After spinning at 17 thousand times G at four degrees celsius for five minutes discard the supernatant and incubate the tubes at 37 degrees celsius on a thermal block completely evaporate the ethanol. Re suspend the pellet with 12 microliters of 10 millimolar tricider chloride at PH 8.5. Next perform a library quality check by diluting the purified and complimentary DNA ten times and using one microliter of the diluted CDNA to run on a bioanalyzer.
Make sure the complimentary DNA product size corresponds to the range of the small RNA in the electropherogram between 136 and 160 base pairs. Calculate the total molarity for each sample. Normalize each sample to 2 nanomolar using the tris hydrochloride at PH 8.5.
Hold the libraries by mixing equal volumes of each two nanomolar sample in a single two hundred microliter PCR tube. Denature and sequence following the steps as described in the manuscript. In this study the library was prepared after RNA isolation and a quality check was performed.
The product size for adapter ligated micro Rnase corresponded to about 136 to 160 base pairs for each sample. The highlighted sections shows the range of the gel that was precisely excised for small RNA library of preparation. This figure shows the gel electrophoresis and electropherograms for the purified complimentary DNA library.
Micro dissected FFPE tissue and serum derived EV's. The representative metrics from a small RNA sequencing run demonstrate the expected cluster density, Q score, cluster passing filter percentage, and the estimated yield and error rates for micro dissected FFP tissue and serum derived EV's. The Q score in both samples showed a value above three indicating 99.9 percent of base call accuracy.
And the error rates were all below one. Each step in library preparation should be carefully executed and tubes should be immediately transferred to ice after each incubation. A critical step in the protocol is cutting the gel precisely to ensure you have an adapter dimer free and pure CDNA library.
Generating CDNA library requires optimization for a high quality sequencing run. Understanding this protocol will help the viewers in extrapolating these minute details to other sequencing library prepration from limited source samples suggest serum and extracellular vesicles. This technique has helped us in identifying new micronase that are associated tumorate aggressiveness and therapy resistance in metastatic prostate cancer patients.
These techniques can extrapolated to other disease types that can aid in Novel biomarker discovery. As this technique uses biological materials such as striation derived serum and tissue samples, one should take appropriate precautions. Further, ethidium bromide is a known carcinogen and should be very carefully handled by using this protocol.
