RT-PCR

Reverse Transcriptase-PCR

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

The following is a great RT-PCR protocol I obtained from David Klein at 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.

Protocol

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

Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1M KCl.
a) Place 100 ul Dynabeads (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.
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.
Resuspend beads in :
2.5 ul Buffer A* (200 mM Tris-HCl, pH 8.3,1.0 M KCl)
2.5 ul Buffer B* (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
* These buffers are supplied with the Retrotherm RT.
Remove 1 ul of reaction. This represents total 32P counts for use in calculating the amount of cDNA synthesized.
Heat at 40 C for 30 min.
Heat at 70 C for 1 hr.
Bind beads and remove all liquid.
Wash beads with 100 ul TE, bind beads, remove liquid.
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.
Please contact me if there are any corrections to be made.
Jim Hutchins
hutchins@umsmed.edu
http://www.umsmed.edu/~hutchins/
Original method from:
From: klein@helix.nih.gov (David Klein)
Subject: Re: Fuzzy balls of mRNA