I have used ACTB and GAPDH as reference gene to normalize qPCR experiments of sperm RNA. since the sperm RNA severely degraded, these genes get amplified in later cycles (>35). i have used cDNA contains 10ng of total RNA/rxn.
The transcripts prm1, together with prm2, codes for protamines which are responsible for packaging the sperm chromatin tightly and is important for protecting the paternal genome, Try these one to see the quality of RNA.
Thanks Dr Ishtiaq Qadri for your suggestion. I agree with you, PRM1 can be used to check sperm RNA quality but I want to do relative expression of some genes using qPCR. for normalizing qPCR experiments i could not use ACTB and GAPDH, because of less abundant and also highly altered expression. In this concern i am looking for SUITABLE reference gene?
Dear Sivashanmugam Parthipan: In studies researchers have used, (β-Actin, ATP synthase subunit beta, Protamine 1, L32 ribosomal protein and Ubiquitin B), of which β-Actin and the L32 Ribosomal protein and they all show the highest stability thus being the most suitable to be considered as reference genes. ATP synthase subunit beta and Ubiquitin B are the other possibilities
Hello Sivashanmugam, i did not work with sperm RNA, but i worked with degraded RNA, i can give you some suggestions:
The expression of all genes will be decreased when your RNA es degraded, even reference genes
One option, design primers again, they should flank in middle part of gene. If you do PCRs do not increase CT values, then you could consider pre-amplification process, some people use this strategy when your transcripts are low abundance.
If you consider that your issue is the reference gene, then you can identify it by yourself using NormFinder or Genorm, these are online tools for identification of reference genes, you can download some sperm data from GEO NCBI (if you have own, it would be perfect) , one example: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE26881. good luck!
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