RNA isolation and reverse transcription are essential steps in gene expression analysis. These techniques allow researchers to extract RNA from cells and convert it into complementary DNA (cDNA) for downstream applications such as PCR, qPCR, and gene expression profiling.
Chloroform, isopropanol, 75% ethanol, TRIzol, RNase-free water or 0.5% SDS (solutions need to be prepared with DEPC-treated water)
a) Homogenization treatment:
| Tissue | Grind the tissue in liquid nitrogen, add 1 mL TRIzol per 50-100 mg of tissue, and use a homogenizer for homogenization. The sample volume should not exceed 10% by volume of TRIzol. |
| Monolayer culture cells | Add TRIzol directly to the culture plate to lyse the cells, add 1 mL per 10 cm2 area (i.e. 3.5 cm diameter culture plate), and pipette several times. The amount of TRIzol should be based on the culture plate area, not on the number of cells. Insufficient TRIzol addition may result in DNA contamination of the extracted RNA. |
| Cell suspension | Collect cells by centrifugation, add 1 mL of TRIzol for every 5-10×106 animal, plant, yeast cells, or 1×107 bacterial cells, and pipette repeatedly. Do not wash cells before adding TRIzol to avoid mRNA degradation. Some yeast and bacterial cells require a homogenizer. |
b) Place the homogenized sample at room temperature (15-30°C) for 5 minutes to completely separate the nucleic acid-protein complexes.
c) Optional step: If the sample contains protein, fat, polysaccharides, or extracellular substances, centrifuge at 10000 × g for 10 minutes at 2-8°C, and take the supernatant. The pellet obtained by centrifugation includes the outer membrane of cells, polysaccharides, high molecular weight DNA, and the supernatant contains RNA. When dealing with adipose tissue, a lot of oil in the upper layer should be removed. To proceed, take the clarified homogenate.
d) Add 0.2 mL of chloroform for every 1 mL of TRIzol used, shake vigorously for 15 seconds, and place at room temperature for 3 minutes.
e) Centrifuge at 10,000 x g for 15 minutes at 2-8°C. The sample is divided into three layers: the bottom layer is a yellow organic phase, the upper layer is a colorless aqueous phase, and an intermediate layer. The RNA is mostly in the aqueous phase, which is approximately 60% of the volume of the TRIzol reagent used.
f) Transfer the aqueous phase to a new tube. If you want to separate DNA and protein, you can keep the organic phase. Precipitate the RNA in the aqueous phase in isopropanol. Add 0.5 mL of isopropanol for every 1 mL of TRIzol used, and leave at room temperature for 10 minutes.
g) Centrifuge at 10,000 × g at 2-8°C for 10 minutes. No RNA precipitation can be seen before centrifugation, but a gelatinous precipitate appears on the side and bottom of the tube after centrifugation. Remove the supernatant.
h) Wash the RNA pellets with 75% ethanol. Add at least 1 mL of 75% ethanol for every 1 mL of TRIzol used. Centrifuge at no more than 7500 × g for 5 minutes at 2-8°C, and discard the supernatant.
i) Place at room temperature to dry or vacuum dry the RNA pellet, and let it dry for about 5-10 minutes. Do not dry by vacuum centrifugation (being too dry will greatly reduce the solubility of RNA).
j) Add 25-200 μL of RNase-free water or 0.5% SDS, pipette several times with a pipette tip, and place at 55-60°C for 10 minutes to dissolve the RNA. If RNA is used for an enzyme digestion reaction, do not use an SDS solution. RNA can also be dissolved in 100% deionized formamide and stored at -70°C.
k) Extracted RNA can be quantified using a spectrophotometer. The spectrophotometer measures the absorbance of RNA at 260 nm, and the purity of RNA can be assessed by the ratio of absorbance at 260 nm and 280 nm (A260/A280). A ratio of 1.8-2.0 indicates pure RNA, while lower ratios indicate contamination with proteins or other contaminants.
a) Each RNA sample (1 µg) will be reverse-transcribed using reverse transcriptase. A separate 1 µg aliquot of the same RNA sample was also used as a no-RT control.
b) For each sample, mix the following:
| DEPC-treated water | 13 µL |
| 5X first strand buffer | 16 µL |
| DTT (0.1 M) | 7 µL |
| Random primer (concentration = 0.1 µg/µL or 1/30 dilution of 3 ug/uL) | 8 µL |
| BSA | 8 µL |
| dNTPs | 3 µL |
| RNase Out | 1 µL |
c) Mix the above reagents (56 µL total volume) by vortexing.
d) Aliquot the above master mix into two 0.5 mL Eppendorf centrifuge tubes, 28 µL each. Add 2 µL of MMLV reverse transcriptase to one centrifuge tube for the RT reaction. Add 2 µL H2O to another centrifuge tube for the no-RT control.
e) Add 10 µL of the above DNase-treated RNA to each centrifuge tube.
f) Pipette to mix.
g) Incubate all samples at 37°C for 1 hour, then at 95°C for 5 minutes to completely inactivate the remaining enzymes, such as MMLV RT and any remaining DNase.
h) Use samples for PCR or store samples at -20°C for use the next day.
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