Method for RT-PCR?

[Jim Hutchins]

Mark Griffiths (BMBMG at biovax.leeds.ac.uk) wrote:
: Does any one have a reliable method for RT-PCR?  Using a kit is an
: option but most kits seem to be extremely expensive. Information
: on methods of RNA purification (kit or otherwise) and source of
: reagents would also be greatly appreciated.

I can't say this method is cheap, but it is reliable. 

I obtained this protocol from Dr. David Klein at NIH (thanks!)

It has worked like a champ in our lab, especially with mRNAs which have
secondary structure.

========

1. Dissolve RNA (30 ug) in 10 ul H20, add 20 ul TE/1 M KCl
2. a. Place 100 ul Dynabeads (Dynal, Inc, Great Neck NY USA) (5 mg/ml) in 
      a 0.5 or 1.5 ml tube
   * You want the Dynabeads coupled to oligo-dT which are sold as part of 
      their mRNA purification kit.  You will also need a magnetic stand 
      capable of holding the tubes (0.5 ml or 1.5 ml) you're using
   b. Bind beads
   c. Remove liquid
   d. Add 100 ul TE/1 M KCl
   e. Wash
   f. Bind beads
   g. Remove liquid

3. a. Add RNA solution to beads
   b. heat to 70 deg C for 2 min and cool slowly to room temperature 
      for 10 min
   c. bind beads
   d. remove liquid

4. Resuspend beads in:
   2.5 ul buffer A (200 mM Tris, 1 M KCl, pH 8.3)**
   2.5 ul buffer B (30 mM MgCl2, 15 mM MgSO4)**
  20   ul dNTPs (2.5 mM each)
   1   ul 32P-dCTP (5 uCi)
   1   ul RNase inhibitor (e.g. RNasin)
   2   ul SuperScript II RT (Gibco-BRL #18064-014)
   5   ul Retrotherm RT (Epicentre Technologies #R19250)
  16   ul H2O

  50   ul total volume
     ** these are supplied with the Retrotherm RT enzyme

5. Remove 1 ul of the reaction.  This represents total counts to use in
calculating cDNA synthesis

6. Incubate 30 min 40 deg C

7. Incubate 1 hr 70 deg C

8. Bind beads and remove liquid

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

10. Resuspend beads in 100 ul TE.  Count 1 ul of this reaction to determine
the amount of cDNA synthesized.  Beads can be used directly in a PCR
reaction (1 ul per rxn).

=======

He gives the following references:

J Biol Chem 269:31969, 1994
Nucl Acids Res 19:4010, 1991
Nucl Acids Res 20:3528, 1992

Also, I think he has a manuscript on this in press, but I do not have the
reference.

I hope this helps.  Contact me if problems, but the credit goes to Dr.
Klein! :-)

Reminder: for names/addresses of suppliers check out Martin Leach's 
  Biotech addresses home page

Disclaimer: I have no connection whatever to the above companies.


--
Jim Hutchins
Assoc Prof Anatomy, Asst Prof Neurology (Research), Univ Mississippi Med Ctr
http://fiona.umsmed.edu/~hutchins/    ***    E-mail:   hutchins at umsmed.edu
``It became necessary to destroy the town in order to save it.''--American
infantry officer firing on Ben Tre, Vietnam, 2/8/68