RNA Extraction and Reverse Transcription Protocol
Licia Miller Product Manager
RNA extraction and reverse transcription experiments are key technologies used in molecular biology to study gene expression and function. RNA extraction is the separation of complete, impurity-free RNA from cells or tissues, while reverse transcription is the conversion of RNA into cDNA for subsequent PCR amplification or sequencing analysis.
This experimental process is of great value in the fields of gene expression analysis, disease diagnosis, drug development, gene function research, and agricultural biotechnology. It can help scientists deeply understand the expression patterns and regulatory mechanisms of different genes under physiological and pathological conditions, and provide important support for life science research and medical applications.
Phase 1 Cell RNA extraction
Steps
1. Cell harvesting: Use at least 10⁶ cells, aspirate the medium, and wash once with 1-2 mL cold PBS.
2. Add TRIzol: After aspirating PBS, add 1 mL TRIzol, gently scrape the test plate to completely lyse the cells, and transfer the TRIzol/cell lysate to a 1.5 mL centrifuge tube.
3. Standing and stratification: Standing at room temperature for 5 minutes, add 250 µL of chloroform, shake vigorously for 15 seconds, and standing at room temperature for 5 minutes.
4. Centrifugation: Centrifuge at 12,000 × g for 15 minutes. Three layers will appear in the centrifuge tube: the upper transparent aqueous phase (containing RNA), the middle white precipitate (DNA), and the bottom pink organic phase.
5. Extract RNA: Carefully transfer the upper aqueous phase to another centrifuge tube, add 550 µL of isopropanol, mix gently, and let stand at room temperature for 5 minutes.
Be careful to leave some aqueous phase (about 1 mm above the DNA layer to prevent DNA contamination).
6. Precipitate RNA: Centrifuge at maximum speed (~12,000 × g) for 20 minutes (up to 30 minutes if yield is low), discard supernatant, add 1 mL 75% ethanol, and centrifuge at 11,500 × g for 5 minutes.
7. Drying and dissolving: Pour off the ethanol and dry the precipitate (avoid over-drying or ethanol residue). When only a very small meniscus of solution remains around the precipitate, add 15-25 µL of DEPC-treated TE buffer or DEPC-treated water to dissolve the RNA.
This is a critical step; if the pellet is over-dried, the RNA will crystallize and be difficult to redissolve. If the ethanol is not evaporated enough, this will also prevent the RNA from going into solution.
8. Dilute the RNA at a ratio of 1:40 (1.2 µl RNA in 48.8 µl TE buffer) into a small EP tube and add it to a micro cuvette. Then measure the absorbance at 260 nm.
The 260/280 ratio should be greater than 1.8. If it is less than about 1.5 - 1.6, the RNA is likely at least partially degraded. Lower ratios can also indicate the presence of DNA or thiocyanate contamination. The RNA concentration (µg/µl) is roughly equivalent to the OD at 260 nm.
Phase 2 DNase treatment
Steps
1. Prepare DNase mixture: Each sample contains 1 µL RQ1 RNase-free DNase, 2 µL DNase 10× Reaction buffer, 6 µL DEPC-treated water, and 0.5 µL RNase Out.
2. Treat RNA: Add 2 µg RNA to a centrifuge tube (calculate the volume based on the RNA concentration determined in the previous stage) , adjust the total volume to 11 µL with DEPC-treated water, and add 9 µL of DNase premix to a total volume of 20 µL.
3. Incubation: Using a thermal cycler, incubate samples at 37°C for 15 minutes, 65°C for 20 minutes, and then place on ice.
4. Centrifugation: Briefly centrifuge each sample to ensure that the sample is concentrated at the bottom of the test tube. The RNA is ready for reverse transcription.
Phase 3 Reverse transcription of RNA
In most cases, each RNA sample (1 µg, or 10 µl from a DNase-treated reaction) will be run with reverse transcriptase, with a second 1 µg aliquot used as a no-RT control.
Steps
1. Prepare the reaction system; Mix the following reagents for each sample (total volume should be 56 µl) and vortex to mix:
- 13 µl DEPC treated water
- 16 µl 5× First Strand Buffer
- 7 µl DTT (0.1 M)
- 8 µl random primers (concentration = 0.1 µg/µl or 1/30 dilution of 3 µg/µl)
- 8 µl BSA
- 3 µl dNTPs
- 1 µl RNase Out
2. Aliquot and react: Aliquot 56 µL of the premix into two centrifuge tubes, one for RT reaction (add 2 µL MMLV reverse transcriptase) and the other for no RT control (add 2 µL H₂O). Add 10 µL DNase-treated RNA to each centrifuge tube and mix well by pipetting.
3. Incubation and inactivation: All samples were incubated at 37°C for 1 hour and then incubated at 95°C for 5 minutes to inactivate the enzyme.
4. Use immediately for PCR or store at -20°C for future use.
For more product details, please visit Aladdin Scientific website.