Reverse Transcriptase-PCR

Reverse Transcriptase-PCR

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

1. Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1M KCl.
2. 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.
   
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 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.
   
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.
    Please contact me if there are any corrections to be made.
    Jim Hutchins
    hutchins@umsmed.edu
    http://www.umsmed.edu/~hutchins/