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Any one can tell me what happens to my RT-PCR? - Urgent !RT-PCR really kills me! (Jun/22/2006 )

Hi every body:

Recently, I run into a strange matter!

The day before yesterday, I Extract Total RNA from human skin which is homogenized in Trizol , then stored in -70 dgree celsius for about 8 months. To my suprise, Agarose gel Electrophoresis shows Total RNA is intact. So I Revesre Transcribe Total RNA later With Superscript II RTase From Life Technology, with which I has ever succeeded in RT the Total RNA into cDNA First strand (the same gene of interest with this time ). when I run PCR with a pair of primers located at the 5’end of the cDNA, I got nothing. Today, While I set a control which template is the truncated cDNA (Seq is same to this first strand of cDNA) got last year, the control runs well. Can any one tell me what happens to my RT-PCR?

Many thanks in advance!

-Cinba-

If the PCR does not work well
try to input more quanity of reverse transcripted RNA.
the reverse transcrition does not work well.

-Najib-

QUOTE (Najib @ Jun 23 2006, 04:33 PM)
If the PCR does not work well
try to input more quanity of reverse transcripted RNA.
the reverse transcrition does not work well.



Hi Najib:

Thanks for your reply!

I later add more Reverse transcribed mRNA to the PCR, about 3 times more, but Nothing appears! Really, it is a headache!

Any more suggestion?

Many Thanks in advance!

-Cinba-

are you sure that your transcript of interest is expressed in these cells?

-aussieuk-

QUOTE (aussieuk @ Jun 23 2006, 06:04 PM)
are you sure that your transcript of interest is expressed in these cells?


Hi aussieuk:

I have Reverse Transcribed the mRNA of Interest successfully. Now I just cannot repeat it.How cumbersome!

-Cinba-

Hi:
How can you confirm you have successful obtain cDNA?
Firstly, you may use OD to detect whether the RT is successful?

-Brainzhang-