To preface, I am new to wet lab. I am infecting six different cancer cell lines with two different virus, and have included mock infections (no virus) as additional controls. After a 12 hour infection, I extract the cell supernatant and fix the cells in the well (for IF). My IF where I stain for np protein show infection in the appropriate groups and no infection in the controls, so I know I'm not somehow accidently adding virus to the mock infections.
I use a viral RNA extraction kit (ezna) where only for the cell supernatants so no cell lysis step. I spray all my tools and bench with DNA and RNA zap before starting, followed by RNaseZap. The kit uses columns, but does not include a DNase step. I consistently get A260/A280 ratio higher than 2.5 (usually 3-3.5) and A260/A230 ratio around between 1-2 and in some cases even as high as 3. I have tried to minimize as much contamination as possible, I am currently trying identify any phenols used during purification. The kit uses carrier RNA and some have suggested that that may skew results. Also my RNA from mock control show similar and sometimes higher amounts of RNA.
When I go on to cDNA synthesis I am not sure of troubleshooting here. It is pretty straightforward protocol we have, although I have now been trying to dilute cDNA (1:10) before qPCR.
I use sybr green kit for qPCR where I ad 2.5 ul of cDNA, 2.5 of primers, and 5 ul of sybr green. I use primers at concentration of 10 uM each. These primers/ the sequence I use is from a senior lab member who has used them reliably. My water controls show appropriate Ct values (high) and I don't see anything indicating primer dimers. My mock infection controls however show Ct values similar to my experimental groups, and sometimes even lower Ct values meaning that something is getting amplified. I use primers for an M1 gene in the virus. Given this information is there any advice on how to troubleshoot and what might be going wrong?
From your description, I don't see that anything is necessarily wrong. It sounds like you are doing a "relative expression" experiment and not an "exact copy number" experiment to look at gene expression. I am assuming that you included a constantly expressed "housekeeping" gene from all of your samples?
Use that as a reference to compare the expression of your gene of interest. It's normal to have variation in Ct values between biological replicates or between treatment groups.
Also, I assume that you included a standard curve to verify that your primers are 95-105% efficient? If not, then you cannot use or publish these data.
Good luck!
In my experience, I analyze the amount of viral genome in supernatants for several days, using a One-step kit with sybr green, in all kits, both one-step and kit to work with DNAc, the concentration of primers that they recommend it goes from 100-300 nM, you can modify this, and see how your results come out, I use 100 nM.
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