Reverse Transcriptase-PCR

This method was submitted by Jim Hutchins from the University of Mississippi Medical Center.

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The following is a great RT- PCR protocol developed by Ignacio Rodriguez at the National Eye Institue, NIH. He gets all the credit; if you have problems with it, contact me :-)
Jim Hutchins
hutchins@umsmed.edu
http://www.umsmed.edu/~hutchins/

 

References

Raineri, I., Moroni, C. and Senn, H.P. (1991).
Improved efficiency for single-sided PCR by creating a reusable pool of first-strand cDNA coupled to a solid phase. Nucleic Acids Res. 19 : 4010-20

Rodriguez, I.R., Mazuruk, K.,Schoen, T.J. and Chader, J.G. (1994). Structural analysis of the human hydroxyindole- O-methyltransferase gene : presence of two distinct promoters. J. Biol. Chem. 269, 31969- 31977.

Rodriguez, I.R. and Chader, G.J., A novel method for the isolation of tissue-specific genes, Nucleic Acids Res., 20 (1992) 3528.

Schoen, T. J., Mazuruk, K., Chader G. J., and Rodriguez, I. R. Isolation of candidate genes for macular degeneration using an improved solid-phase subtractive technique. Biochem. Biophys. Res. Commun., 213 (1995) 181-8.

 

Protocol

PROCEDURE FOR cDNA SYNTHESIS ON DYNABEADS oligo(dT)₂₅ (5/4/94)

1. Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1M KCl.

2. a) Place 100 ul Dynabeads oligo(dT)₂₅ (5 mg/ml) in a 0.5 ml tube.
b) Bind beads.
c) Remove liquid.
d) Add 100 ul TE/1M KCl.
e) Wash.
f) Bind beads.
g) Remove liquid.

3. a) Add RNA to beads.
b) Heat to 70 C for 2 min and cool slowly to RT for 10 min.
c) Bind beads.
d) Remove liquid.

4. Resuspend beads in :
2.5 ul Buffer undefined (200 mM Tris-HCl, pH 8.3,1.0 M KCl)
2.5 ul Buffer undefined (30 mM MgCl2 and 15 mM MnSO4)
20.0 ul dNTPs (2.5 mM each)
1.0 ul 32P-dCTP (5 uCi)
1.0 ul RNasin-Pharmacia
2.0 ul SuperScript II RT (200 U/ul)(Gibco BRL # 18064-014)
5.0 ul Retrotherm RT (1 U/ul) (Epicentre Technologies # R19250)
16.0 ul H20
-----------------
50 ul

5. Remove 1 ul of reaction. This represents total 32P counts for use in calculating the amount of cDNA synthesized.

6 . Heat at 40 C for 30 min.

7. Heat at 70 C for 1 hr.

8. Bind beads and remove all liquid.

9. Wash beads with 100 ul TE, bind beads, remove liquid.

10. Resuspend beads in 100 ul TE. Count 1 ul of beads to calculate the amount of cDNA synthesized. Use 1 ul of beads per PCR .

I think the real secret here is the removal of secondary structure by the thermostable RT coupled with the immobilized RNA forming a "reusable" pool of template. This works well with total RNA or a poly(A) fraction.