Begin by performing the MM QPCR assay for telomere length measurement. Then open the software and select the plate setup feature. After that, choose the view or edit plate option from the dropdown menu.
Now highlight all the wells on the plate. Click select floraphores. Check the box labeled cyber, and ensure all other boxes are unchecked.
Then click okay to confirm. While maintaining the highlight on all wells, locate and check the box next to cyber, adjacent to the word load to label all the wells with cyber. Now highlight the three non template control wells positioned either at the top or bottom of the standard control serial dilution.
Then from the sample type menu on the right, select NTC. Highlight the 21 wells that make up the standard control serial dilution, and choose standard from the sample type menu. While these wells are still selected, click on technical replicate, in the replicate size menu, choose three.
Then select horizontal and click apply. After that, scroll down to the dilution series section and enter two in the dilution factor field. For the P1 plate, input two times 10 to the power of negative three in the starting concentration field.
Then select the box for decreasing and click apply to confirm the settings. For the P2 plate, enter 3.13 times 10 to the power of negative five in the starting concentration field. Ensure to check the box for increasing and then select apply to finalize the setup.
Now highlight the columns corresponding to PCR strip A from the sample type menu, choose unknown, then proceed to select technical replicate. In the replicate size menu, pick three and opt for horizontal before clicking apply. Click okay at the bottom right of the plate editor window.
Confirm the changes by clicking yes when prompted. Next, verify that the curves in the quantification tab appear appropriate for both step nine and step 12, and that the efficiency values between these two steps do not diverge by more than 10%For P1, select step nine and highlight the 21 wells corresponding to the standard curve. Copy the CQ values from the lower right corner of the software window.
Then open the Telomere data sheet template and paste these values into the standard one sheet column B.Afterward, input the slope value of step nine into cell C four in the standard one sheet, check that the coefficient of variation for each standard dilution triplicate is below 0.1. On CFX, exclude any outlier data points from the analysis that make a set of triplicates fall out of range. Now, confirm that only the 72 sample wells in the P1 analysis file are highlighted in blue in the quantification tab.
Then in step nine, copy the CQ and SQ values found in the lower right corner of the software window and paste them into the samples P1 sheet beginning at cell D3.Using CFX maestro, ensure all CQ values for analytical samples are within the range of the lowest and highest CQ values of the standard curve. In the Excel sheet, verify that the starting concentration in the NTC is less than 5%of the average amount of DNA in the sample wells. For sample level quality control, examine the standard deviations and coefficient of variations in columns J and K on the samples P1 and P2 sheets.
Verify that the individual sample interplate CVs in the P1 versus P2 sheet, specifically in column G, do not exceed 0.05. If a sample's interplate CV is higher than acceptable, remove up to one triplicate from the calculation for each sample. For plate level quality control, check that the average interplate variation across all samples on each plate is less than 0.05.
If a sample's interplate CV is higher than acceptable, remove up to one triplicate from the calculation for each sample. For additional plate level quality control, check that the overall interplate variation in P1 versus P2 sheet is less than 0.06.
