Strange thing about RNA concentration and real-time melting curve - (May/18/2006 )
After trizol extraction and RNA purification. I freezed down the samples at -20 and thawed them out several hours later to test concentration by spectrophotometer. I got 120.5ng/ul.
I freezed down the samples again at -80 and before doing cDNA synthesis in the next day, I double checked the concentration and it became 58ng/ul. I was wondering what happened to my RNA yesterday to make it so much less.
I assumed the 58ng/ul was the correct concentration and continued cDNA synthese with 0.7ug RNA. After I run the cDNA in real-time PCR, I got two melting peaks, one is about 83.5oC, the other one is 86oC; they looks more like one wide peak rather than two peaks in the actual melting curve picture. This melting curve pattern never happened with my primer and my gene before, I was totally lost about what's going on!?
Any comments?
DNA contamination in RNA sample? PCR product of different sizes (one with intron probably while the other just cDNA seq), thus the two peaks.
Since primers worked well before in your previous real time PCR, shouldn't be the following problems: mispriming and primer-dimer artifacts.
As for the inconsistent reading of RNA conc....
If you're using cuvettes to measure the sample, do try to use the same cuvette for setting blank reference and also sample reading. Changing of cuvettes, especially those NOT made of quartz material will sometimes change the reading. I use quartz capillary tube to read my RNA now, but used to read with Eppendorf's spectrophotometer.
Dear Isek,
1. Repeat freezing and thawing of sample will degrate your RNA. Try to avoid that.
2. Did you see a signifcant positive signal from your amplification curve? What is the Ct value?
If you doubt with you real-time result, ry to run on gel. If you have two peaks from the melting
curve, usually you will see a specific band aong with someting else (could be primer dimer or
unspecific and).
Regards
Thanks for all of the comments.
1. Repeat freezing and thawing of sample will degrate your RNA. Try to avoid that.
2. Did you see a signifcant positive signal from your amplification curve? What is the Ct value?
If you doubt with you real-time result, ry to run on gel. If you have two peaks from the melting
curve, usually you will see a specific band aong with someting else (could be primer dimer or
unspecific and).
Regards
I did see a signifcant positive signal from amplification curve. I checked my pcr product on a gel. And I got bands much fainter that DNA ladder. All my pcr product showed one band with expected size, but with a bit smears.
Any other idea about what's going on?
Things in molecular biology are just soooooooooo strange.
Today I run 3 cDNA templates. One from previous experiment that works and two from last pcr that doesn't work. All conditions are exactly the same. They all worked today and have nice and nomal melting curve as I usually got.
I still don't know what's going on, but the pcr seems OK now. Maybe the real-time machine's been a bit stupid last week.