This protocol utilizes total RNA because if 18SrRNA is used as an internal control, RNA with poly(A) cannot be used in subsequent experiments. and the RNA must be treated with DNAzyme to remove all contaminating genomic DNA. this experiment was derived from PCR Laboratory Guide (2nd ed.) by Seedkang, Qu Lijia.

Operation method

Single-stranded cDNA synthesis

Move

I. Materials

1. Buffers, solutions and reagents

Double-distilled water for RNAase removal

10X RT-PCR buffer (double-distilled water for RNAase removal, 100 mmol/L Tris-HCl, pH 8.3, 500 mmol/L KC1, 15 mmol/L MgCl₂ )

2. Enzyme and enzyme buffer

MMLV reverse transcriptase (100~200U/ul)

SUPERaseINRNA enzyme inhibitor (20U/ul;Ambion)

3. Nucleic acids and oligonucleotides

dNTP mixture (10 mmol/L, contains all four dNTPs)

Random primers (5umol/L)

Total RNA (up to 2.5ug)

4. Experimental equipment

Thin-walled PCR tubes

II. Methods

1. Add 2ul of 50umol/L random primers on ice to thin-walled PCR tubes (one reaction per tube).

2. Add 2.5ug of total RNA.

3. Add 4ul of 10 mmol/LdNTP mixture.

4. Add RNAase-removing double-distilled water ( _{RNaSe-freeH20} ) to a volume of 16 ul.

5. Heat at 70°C for 10 min to denature the secondary structure, then immediately place on ice.

6. Add 2ul of 10XRT-PCR buffer.

7. Add 1ul of 20U/ul SUPERaselN.

8. Add 1ul of 100~200U/ul MMLV Reverse Transcriptase.

9. Temperature bath at 42°C for 1h.

10. Heat at 95°C for 10min to inactivate reverse transcriptase.

11. Continue amplification step or stop the reaction and store at -2°C.

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