Material

1. randon primer 1㎕
2. 2MUG RNA F10 1.95㎕
3. 2MUG RNA m1 1.95㎕
4. 10mM dNTP 1㎕
5. ddH₂O
6. 5× FSB 4㎕
7. 0.1M DTT 2㎕
8. RNase out 1㎕
9. M-MLV RT 1㎕
10. eppendorf

Procedure

A 20-㎕ reaction volume can be used for 1 ng-5MUG of total RNA or 1-500 ng of mRNA.

1. Add the following components to a nuclease-free microcentrifuge tube:
   • 1㎕ oligo(dT)_12-18(500μg/ml), or 50-250 ng random primers, or 2pmole gene -specific primer
   • 1 ng to 5MUG total RNA or 1 ng to 500ng of mRNA
   • 1㎕ 10 mM dNTP Mix(10 mM each dATP,dGTP,dCTP and dTTP at neutral pH)
   • Sterile, distilled water to 12㎕
2. Heat mixture to 65℃ for 5 min an quick chill on ice. Collect the contents of the tube by brief centrifugation and add:
   • 4㎕ 5X First-Strand Buffer
   • 2㎕ 0.1 M DTT
   • 1㎕ RNaseOUTTM Recombinant Ribonuclease Inhibitor (40 units/㎕)

   (Note: When using less than 50 ng of starting RNA, the addition of RNaseOUTTM is essential.)

3. Mix contents of the tube gently and incubate at 37℃ for 2 min.
4. Add 1㎕(200 units) of M-MLV RT,a and mix by pipetting gently up and down. If using random primers, incubate tube at 25℃ for 10 min.
5. Incubate 50 min at 37℃.
6. Inactivate the reaction by heating at 70℃ for 15 min.

Result

Preparing Samples

Homogenizing samples

• Determine your sample type, and perform homogenization at room temperature according to the table below. The sample volume should not exceed 10% of the volume of TRIzol Reagent used for homogenization. Be sure to use the indicated amount of TRIzol Reagent, because an insufficient volume can result in DNA contamination of isolated RNA.

Sample Type	Action
Tissues	Add 1mL TRIzol Reagent per 50-100 mg of tissue sample. Homogenize sample using a glass-Teflon or power homogenizer. Note: Process or freeze tissue samples immediately upon collection.

RNA Isolation Procedurec

Always use the appropriate precautions to avoid RNase contamination when preparing and handling RNA.

RNA precipitation

1. (Optional) When precipitating RNA from small sample quantities (<10⁶ cells or < 10 mg tissue), add 5-10μg of RNase-free glycogen as a carrier to the aqueous phase.
   • Note: Glycogen is co-precipitated with the RNA,but does not inhibit first-strand synthesis at concentrations ≤4mg/mL,and does not inhibit PCR.
2. Add 0.5mL of 100% isopropanol to the aqueous phase, per 1mL of TRIzol Reagent used for homogenization.
3. Incubate at room temperature for 10 minutes.
4. Centrifuge at 12,000 × g for 10 minutes at 4℃.
   • Note: The RNA is often invisible prior to centrifugation, and forms a gel-like pellet on the side and bottom of the tube.
5. Proceed to RNA wash.

Proceed to RNA wash.

1. Remove the supernatant from the tube,leaving only the RNA pellet.
2. Wash the pellet,with 1mL of 75% ethanol per 1mL of TRIzol Reagent used in the initial homogenization.
   • Note: The RNA can be stored in 75% ethanol at least 1 year at-20℃, or at least 1 week at 4℃.
3. Vortex the sample briefly,then centrifuge the tube at 7500 × g for 5 minutes at 4℃.Discard the wash.
4. Vacuum or air dry the RNA pellet for 5-10 minutes. Do not dry the pellet by vacuum centrifuge.
   Note: Do not allow the RNA to dry completely,because the pellet can lose solubility.Partially dissolved RNA samples have an A_260/280 ratio<1.6.
5. Proceed to RNA resuspension.

RNA resuspension

1. Resuspend the RNA pellet in RNase-free water or 0.5% SDS solution(20-50㎕) by passing the solution up and down several times through a pipette tip.
   • Note: Do not dissolve the RNA in 0.5% SDS if it is to be used in subsequent enzymatic reactions.
2. Incubate in a water bath or heat block set at 55-60℃ for 10-15 minutes.
3. Proceed to downstream application, or store at -70℃.

This week, we did transformation.

We transform B0034, B0015, I765001, I732005.