Small RNA extraction ●TIMING 1 h
1| Prepare RNA from fresh or cry preserved tissue or cells (or cell lines) with the use of a mirVana miRNA isolation kit, following the manufacturer’s instructions and using the small-RNA fraction option, which allows purification of small RNA molecules of 200 nt. A 50 μl volume is obtained.
2| Precipitate the RNA from the 50 μl volume of sample obtained in Step 1 by adding 2.5 μl of 5 M NaCl and 150 μl ethanol, keep it on dry ice for 30 min, centrifuge it at 14,000 g for 20 min at 4 °C, discard the supernatant and rinse the precipitate with 80 % ethanol before allowing it to air dry. Dissolve the RNA precipitate in 5 μl DEPC water. If the yield of RNA is thought to be >0.5 μg, determine the RNA concentration by measurement of absorbance at 260 nm. If the concentration of RNA is >1μg μl–1, then it should be adjusted to 1μg μl–1 with DEPC water.
▲CRITICAL STEP Precipitation of a small amount of small RNAs requires that the volume of the solution be small.
■PAUSE POINT The RNA solution can be stored at –80 °C for several weeks.
Polyadenylation of small RNAs●TIMING 2h
3| In a sterile 0.2 ml microfuge tube(Table 2).
4| Incubate the mixture at 37 °C for 1h.
■PAUSE POINT The incubation can be extended up to 16 h.
5| Add 50 μl phenol-chloroform to the reaction mixture. Mix, then spin at 13,000 g at 4 °C for 15 min.
6| Collect the aqueous phase, add 5 μl (1/10 vol), 3 M sodium acetate (pH 5.2), add 150 μl (3 vol) 100 % ethanol, and add 1 μl glycogen (20 μg/ μl). Precipitate Poly (A)-tailed small RNA for at least 2 h at –20 °C. Precipitation overnight is preferred.
7| Spin precipitated Poly (A)-tailed small RNA at 13,000 g for 20 min.
8| Carefully remove the supernatant and add 1ml 80 % ethanol to wash the pellet. Spin at 12,000 g at 4ºC for 10 min.
9| Carefully remove all the liquid, avoiding touching the pellet. Air–dry the pellet at room temperature.
10| Dissolve the dried pellet in 10 µl DEPC-H2O.
■PAUSE POINT The sample can be stored at–20 °C for several months.
Ligation of 5’adaptor to Poly (A)-tailed small RNA ●TIMING 1.5 h
11| In a sterile 0.2 mL microfuge tube(Table 3).
12| Incubate the mixture at 37 °C for 1h.
■PAUSE POINT The incubation can be extended up to 16 h.
13| Add 50 μl phenol-chloroform to the reaction mixture. Mix, then spin at 13,000 g at 4 °C for 15 min.
14| Collect the aqueous phase, add 4 μl (1/10 vol), 3 M sodium acetate (pH 5.2), add 120 μl (3 vol) 100 % ethanol, and add 1 μl glycogen (20 μg/ μl). Precipitate 5’adaptor-small RNA-Poly (A)n for at least 2 h at –20 °C. Precipitation overnight is preferred.
15| Spin precipitated 5’ adaptor-small RNA-Poly (A)n at 13,000g for 20 min.
16| Carefully remove the supernatant and add 1ml 80 % ethanol to wash the pellet. Spin at 12,000 g at 4ºC for 10 min.
17| Carefully remove all the liquid, avoiding touching the pellet. Air–dry the pellet at room temperature.
18| Dissolve the dried pellet in 10 µl DEPC-H2O.
■PAUSE POINT The sample can be stored at–80 °C for several weeks.
Reverse transcription ●TIMING 1.5 h
19| Combine the following in a 0.2 ml microfuge tube on ice (Table 4).
20| Incubate at 65°C for 5 min, then place on ice for 2 min.
21| Prepare the following cDNA Synthesis Mix, adding each component in the indicated order (Table 5).
22| Add 10 µl of cDNA Synthesis Mix to each RNA/primer mixture, mix gently, and collect by brief centrifugation.
23| Incubate the mixture at 50 °C for 1 h.
24| Terminate the reactions at 85°C for 5 min then chill on ice.
25| Collect the reactions by brief centrifugation. Add 1 µl of RNase H to each tube and incubate for 20 min at 37°C.
■PAUSE POINT The sample can be stored at–20 °C for several months.
PCR amplification of small RNA-derived cDNAs by 5’miR-RACE and 3’miR-RACE
●TIMING 4.5 h
First-round amplification ●TIMING 2.5 h
26| In a sterile, 0.2 ml microfuge tube, mix the following reagents for each experimental and control sample from Step 25 (Table 6).
27| Add a 1 ml aliquot of the diluted first-round amplification products (obtained in Step 25 above) and 25 pmol each of mirRacer 3’ primer and mirRacer 5’ primer (Fig. 1b). Set up a ‘no-template’ control as well.
28| Mix and then heat in a DNA thermal cycler for 5 min at 98 °C to denature the first-strand products and to activate the polymerase.
29| Carry out 25 cycles of amplification with a ‘step’ program (Table 7).
■PAUSE POINT The products can be stored at -20 °C for several weeks.
Second-round amplification ●TIMING 2.5 h
30| Dilute a portion of the amplification products from the first round 1: 20 in TE buffer.
▲CRITICAL STEP A second round of amplification is required because the use of mirRacer 5’ primer and the mirRacer 3’ primer (in combination with a universal primer that binds to all of the cDNA templates present in the starting mixture) results in a substantial yield of a pool of non-gene-specific products. The second round, which uses a gene-specific primer (again in combination with a universal primer), eliminates most or all of the nonspecific products.
31| In a sterile, 0.2 ml microfuge tube, mix the following reagents on ice (Table 8).:
32| Add a 1 ml aliquot of the diluted first-round amplification products (obtained in Step 31 above) and 25 pmol each of primers GSP2 and GSP1 (Fig. 1). Set up a ‘no-template’ control as well.
■PAUSE POINT The PCR products can be stored at –20 °C for several months.
33| Mix and heat in a DNA thermal cycler for 5 min at 98 °C to denature the first-strand products and to activate the polymerase.
34| Carry out 30 cycles of amplification with a ‘step’ program (Table 9).
PAUSE POINT The products can be stored at -20 °C for several weeks.
35| Separate 10 % of the products of first-and second-round amplification by electrophoresis through a 2.5 % agarose gel. Use the information gained from this analysis to optimize the miR-RACE procedure (see ANTICIPATED RESULTS).
■PAUSE POINT The DNA solution can be stored at–20 °C for several months.