Feb 16, 2025
QPCR Protocol
- 1Caltech
Protocol Citation: Yujie Fan, Chelsie Steele 2025. QPCR Protocol . protocols.io https://dx.doi.org/10.17504/protocols.io.ewov1d79pvr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 13, 2025
Last Modified: February 16, 2025
Protocol Integer ID: 118269
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Abstract
Collecting gene data from various cells - seeing what sort of gene is being expressed
Materials
Cell Collection
Cell Collection
Remove cells from the incubator. This protocol is for a 24-well plate.
Aspirate the cell media and wash the cells gently once with DPBS.
Aspirate the DPBS and add 350ul Trizol (Cat No.R2051, Zymo Research) onto the cells directly. Increase the volume of trizol if you have a well plate larger than a 24 well plate.
Use P1000 to pipette up and down to collect all the cell lysis. Make sure to go through all the corners The bottom should become clear if all cells have been detached. At this point, the cell lysis can be frozen down in 1.5mL Eppendorf tubes at -80°C, and stored up to 3 days.
RNA extraction from cells
RNA extraction from cells
Once the cell lysis has been collected in 1.5mL Eppendorf tubes, the rest of the RNA extraction is preformed using the Direct-zol RNA Miniprep kit (Cat No. R2051) and protocol provided by Zymo Research.
Use a P1000 pipette to add an equal volume (350uL) of 100% ethanol to each sample. Pipette up and down multiple times to thoroughly mix the ethanol and trizol, and transfer the entire mixture into a Zymo-Spin IICR column. Centrifuge the columns at x10,000g for 1 minute at room temperature. In the meantime, take out DNAse 1 to thaw.
Transfer the columns into a new collection tube, and discard the flow-through. Add 400uL of RNA Wash Buffer (with ethanol already added) to each column, and centrifuge at x10,000g for 1 minute at room temperature.
In a separate 1.5mL Eppendorf tube, mix 5uL of DNase 1 with 75uL of DNA digestion buffer (per sample). Mix by gentle inversion/pipetting, and add 80uL of this mixture to each sample column. Incubate for 15-20 minutes at room temperature.
After incubation, add 400uL RNA Pre-Wash Buffer to each sample, and centrifuge at same conditions. Discard the flow through.
Add 700uL of RNA Wash buffer to each column and centrifuge again at same conditions. Discard the flow-through and centrifuge again to ensure complete removal of ethanol and washing buffer.
Transfer the columns to pre-labeled RNAse-free Eppendorf tubes. Add 50uL of DNAse/RNAse-free water to each column, and let sit for one minute.
Centrifuge at same conditions for one minute. Discard flow-through and Nanodrop the RNA concentrations. At this point, RNA can be stored long-term in -80°C.
CDNA Processing
CDNA Processing
Calculate volume of RNA and UPH2O required for cDNA synthesis. In this, protocol we use 1000ng of RNA per sample.
| A | B | C | D | E | F | |
| name of cell | RNA CONCENTRATION | 1000/rna concentration = CDNA | total volume - superscript volume - cdna = water | 5X SUPERSCRIPT | TOTAL VOLUME | |
| 2037 | 126.6 | 7.9 | 24.1 | 8uL | 40uL | |
| 2052 | 88.7 | 11.3 | 20.7 | 8uL | 40uL |
Example calculation for a total of 40uL of cDNA.
Add calculated volumes of UPH2O and RNA to PCR tubes and add 8µL (or 1:5 ratio compared to total volume) of SuperScript‱ VILO‱ MasterMix to each reaction (40µL final vol). Vortex and spin down the PCR tubes.
Transfer the PCR tubes to a thermocycler, and run under the following conditions.
| 25°C | 10 min | |
| 42°C | 60 min | |
| 85°C | 5 min | |
| 4°C | Hold |
THERMOCYCLER SETTINGS
Wait until the run has finished and then proceed to the next section (75 minutes).
QPCR
QPCR
16m 15s
16m 15s
Once the cycling has finished, transfer the 40uL cDNA from each sample to a new Eppendorf tube. Calculate how much water must be added to each sample tube. Each well needs 5uL of water + 1uL of cDNA (6uL total), and so if you have 15 genes per sample, each sample needs 15 genes x 3 replicates = 45 wells (so around 45uL of cDNA + 225uL of water). It is recommended to make extra (for instance, 50 wells in this case). Make sure to thoroughly pipette the water and cDNA mixture, and then disperse 6uL of the mixture into each well using a repeating pipette.
| A | B | C | D | |
| 18 (round up to 22) | 25 ul | 107 ul | 22 x 6 = 132 | |
| 48 ( round up to 55) | 75 ul | 255 ul | 55 x 6 = 330 | |
Example of cDNA calculation for QPCR
In another separate tube, prepare the SYBER Green and primer mixture. You need a separate Eppendorf tube for each gene you are looking at. Again, note that you want to prepare for more wells then needed. For example, if you have 8 samples, you will need 8 samples x 3 replicates = 24 wells per gene, so it recommended to prepare for 30 wells. For each gene tube, add 7.5uL of SYBER Green with 1.5uL of Primer per well. So, for 30 wells, this would be 225uL SYBER Green + 45uL of Primer for each gene tube. Make sure to thoroughly pipette the SYBER Green and primer mixture, and then disperse 9uL of the mixture into each well using a repeating pipette.
| A | B | C | D | E | F | G | H | I | J | K | L | |
| cell sample 1 + gene 1 | cell sample 2 + gene 1 | cell sample 3 + gene 1 | cell sample 4 + gene 1 | cell sample 5 + gene 1 | ||||||||
| cell sample 1+ gene 1 | cell sample 2 + gene 1 | cell sample 3 + gene 1 | cell sample 4 + gene 1 | cell sample 5 + gene 1 | ||||||||
| cell sample 1 + gene 1 | cell sample 2 + gene 1 | cell sample 3 + gene 1 | cell sample 4+ gene 1 | cell sample 5 + gene 1 | ||||||||
| cell sample 1+ gene 2 | cell sample 2 + gene 2 | cell sample 3 + gene 2 | cell sample 4 + gene 2 | cell sample 5 + gene 2 | ||||||||
| cell sample 1 + gene 2 | cell sample 2 + gene 2 | cell sample 3 + gene 2 | cell sample 4 + gene 2 | cell sample 5 + gene 2 | ||||||||
| cell sample 1 + gene 2 | cell sample 2 + gene 2 | cell sample 3 + gene 2 | cell sample 4 + gene 2 | cell sample 5 + gene 2 |
Example QPCR plate
After the plate is all finished, add the BioRad microseal to the top of the plate. Line up the dashed lines to be at the edges to ensure that all of the wells are covered. Seal the plate tightly, and then spin down the plate in a PCR plate spinner, ensuring that the wells are pointing inwards.
Transfer the prepared plate to a QPCR machine (we used CFX Opus 384). Input the well plate, and choose the relevant protocol. Our protocol was the following since we used SYBER green master mix:
95 degrees for 10 minutes
10m
95 degrees for 15 seconds
15s
58 degrees for 1 minute
1m
Plate Read
Go to step 2 39 times
65-95 degree gradient for 5 minutes at a rate of 0.5 degrees celsius/cycle
5m
Plate Read
Then plate is kept at 4 degree until you are ready to remove it
When machine is finished running, the data can be exported to a USB for further analysis.
QPCR ANALYSIS
QPCR ANALYSIS
The machine will give an output of the qPCR data. Copy the relevant rows and transpose them in excel.
Line up each data in the same way the well plate was prepared, you should end up with a matrix which exactly matches the well plate inputs.
Check each set of 3 replicates and check to ensure there are no outliers. if there are Δ > 1.00 then this data point must be ignored.
Average the data to get one number per gene per sample.
Use the formula antilog (average # of gene A - average # of housekeeping (control)) note that housekeeping after this formula should always equal 1.000.