I want to know whether the RNA i have isolated from Klebsiella pneumoniae is in good condition. I would also like to know what the four bands are (there should be only 3 bands and 5s is not visible so their should be only two, i believe the 2 light bands should be 23 and 16s what are the other two bands ). I facing some problem in cDNA conversion, that's why i want to to know whether my RNA is good. I used qiagen kit for extraction.
Lane 1 ladder 100bp (100bp - 1kb)
lane 2,3 Rna without Dnase treatment
lane 4,5,6,7 after Dnase treatment.
Thank You.
your RNA looks good, banding is good too, How do you do cDNA synthesis, the sheering below the lowest band inturn shows prresence of mRNA too, use highscript reverse transcription kit for large amount of cDNA or if you r using omniscript or superscript please maintain the template concentration of Total RNA as 1 microgram
Thank you Sandeep,
But what are the 4 bands present?
Iam using QuantiTech from qiagen and my total rna was below 1ug. But my qpcr didn't pick any thing even at higher concentration of cdna sample.
RNA looks good and hope in Bioanalyzer RIN value will be also good too.For cDNA superscript III kit from Fermentus does give well results, so, just try once.
Thank you very much David. Is it not possible to check the integrity of RNA without denaturing gel. My spec reading show it is a good RNA 260/280 = 2.2 . And by the way it is normal agarose gel.
what type for qpcr work u r doing standard ya relative quant, after making cDNA did u dilute it with 1: 5 ratio with nuclease free water then use it as template for qPCR, u should get results...
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