I am optimizing real time primers for 10 genes in normal pcr along with GAPDH. Two genes got amplification (GAPDH too) and remaining dosen't. i was using diluted cDNA at the rate of 1:10 as template. All the genes am optimizing was already published one with the same cell line and i was using my own primer but not the published one. kindly help me to optimize all the genes.
Hi! I have some suggesstions base on my experience.
1-Please make sure about RNA purity. It will have a direct effct on real-time results. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of RNA. A ratio of ~2.0 is generally accepted as “pure” for RNA.
2- Please make sur that for cDNA synthesis, enough RNA concentration is used as start material. Some kits suggest 500ng RNA and integrity and reen number is suggested in their product booklet. Some times RNA molecules are broken dyring extractions. in that case you need to add more RNA template as starting material.
3- please check the cDNA in normal pcr and use a tested primer that previously it was working to make sure that the cDNA is working.
4- Please check the PCR running condition and avoid any contaminations.
5- Please check your primers with other tested cDNA. be sure that the primers are working.
Hope it will help you. Good luck.
Hai Maureen, we used 800ng of RNA to make 20 ul of cDNA reaction. From the cDNA 2ul was used for 25 ul of pcr reaction. we got amplification at 18th cycle of real time pcr (GAPDH).
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