Optimized Welsh DDRT-PCR Protocol

MATERIALS:

Special equipment:

 

1. Thermocycler for PCR, such as the Perkin-Elmer (Norwalk, CT) oil-free 9600 GeneAmp PCR system
2. Sequencing apparatus such as Sequi-gen sequencing cells from Bio-Rad (Hercules, CA)
3. Gel dryer (like the Bio-Rad model 583).

Chemicals and reagents (listed in order of need):

 

1. RNAzol ^TM B from BIOTECX Laboratories (Houston,TX), Microcarrier ^TM from Molecular Research Center, Inc. (Cincinnati, OH).
2. Reverse transcriptase (RT) buffer from GIBCOBRL (Gaithersburg, MD), deoxynucleotide triphosphate mixture (dNTPs) from Pharmacia Biotech (Piscataway, NJ), dithiothreitol (DTT, GIBCOBRL), RNase inhibitor , Boehringer (Mannheim), SuperscriptII reverse transcriptase , (GIBCOBRL).
3. Taq DNA polymerase and buffer, (Boehringer), a [ ³² P]-dATP (3000Ci/mmol), Amersham International (Amersham).
4. Loading buffer (90% deionized formamide, 10mM EDTA , pH 8.0, 0.025% bromophenol blue and 0.025% xylene cyanol), acrylamide, bis-acrylamide, urea (molecular biology grade), Whatmann 3MM paper , Tracker Tape ^TM (Amersham International), X-Ray film s.
5. Ethanol , Na Acetate .
6. TA-Cloning Kit from Invitrogen (San Diego, CA).
7. "Hot Tub" cycle sequencing kit (Amersham International), STET buffer (8% w/v sucrose, 0.1% w/w Triton X-100, 50mM EDTA , pH 8.0, 50mM Tris-HCl, pH 8.0), lysozyme, cetrimide (CTAB), agarose.

DETAILED PROCEDURE:

RNA preparation

Any type of RNA preparation may be used if the RNA obtained is undegraded. RNA extraction with RNAzol ^TM B is rapid (2 hours) and simple to perform. The expected yield is 1-1.5ug/mg brain tissue and 10ug/mg of cultured cells (aprox. 10 ⁷ cells). The yield can be improved by adding Microcarrier ^TM to the homogenization step. In order to overcome minor changes arising from differences between individuals, it is very important to pool tissue samples from at least three experimental animals. Special care should be taken to avoid RNA degradation, thus the homogenization step should be carried out rapidly and external RNase sources must be avoided. Other RNA extraction procedures such as the guanidinium thiocyanate-cesium chloride method2 or cytoplasmic RNA extraction4 are also recommended. Poly (A)+ mRNA purification from the total or cytoplasmic RNA can also improve the results, as amplification of mitochondrial RNA or of partially degraded transcripts is avoided. However, It was noted by some researchers that the use of mRNA can cause problems in the DD reaction, since poly dT can carry over into the PCR step.

Differential Display

I. First strand cDNA synthesis. In this step the RNA is reverse transcribed using a primer whose sequence is arbitrarily chosen. The enzyme reverse transcriptase will extend those primers which have annealed to RNA molecules to yield single stranded cDNAs. Incubate the RNA samples at 65C for 10 min and transfer immediately to ice. Reaction mixture.

 

				Stock solutions     Final concentration
				2ul 5X RT buffer    1X
				1ul 100mM dNTPs     10mM
				1ul 100mM
				DTT
				10mM
				1ul 10uM oligodeoxynucleotide (17-mer~undefined  1uM
				0.25ul RNase inhibitor (40U/ul)   1U/ul
				0.25ul SuperscriptII RT (200U/ul)  5U/ul
				1ul RNA (0.5ug/ul)    50ng/ul
				DDW (double distilled water) to 10ul