Lentivirus titration using qRT-PCR
Reagents:
- lenti-X qRT-PCR titration kit from Clontech, Cat: 631235.
To titrate lentivirus titers, the qRT-PCR approach can be used which is easy, reliable and fast. I use the kit from Clontech and follow the protocol.
1. Prepare aliquots of lentivirus of 150 ul.
2. Isolate RNA from lentivirus using the included \'Viral RNA isolation kit\'.
3. Elute RNA with 50 ul RNase-free H2O.
4. Remove plasmid DNA using the provided DNase from the titration kit.
5. Prepare serial dilution of RNA samples and perform qRT-PCR as recommended.
6. When using Roche lightcycler 480 to do the qRT-PCR, use the LMP dye.
7. To calculate the titers (TU/ml), use the formula: (copy number)*10*2*50/150/2.
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SIRT6 links glucose and lipid metabolism
SIRT1, a histone deacetylase, has drawn considerable attention due to its important roles in metabolic regulation and longevity. SIRT1 is induced by fasting and suppressed by feeding. SIRT1 increases glucose production and beta oxidation in liver to meet energy needs during food deprivation. Another member of SIRT family, SIRT6 follows similar expression patterns as SIRT1. The function of SIRT6 in metabolic regulation remains unknown.
Kim and colleagues found SIRT1 can regulate SIRT6 expression in liver. Specifically, SIRT1 functions together with FOXO3a and stimulates SIRT6 transcription during fasting. The activation of SIRT6 directly suppresses gene expression of metabolic enzymes involved in triglyceride synthesis and glycolysis in liver. Deficiency of SIRT6 causes increases of glucose utilization, reduced beta oxidation and as a result, fatty liver. More importantly, Kim et al found the expression of SIRT6 is decreased in human fatty liver samples which indicates SIRT6 may play critical roles in liver steatosis in clinical settings. This study landed in Cell Metabolism of this month.
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