Dec 18, 2023
Version 1
Quantitative real-time PCR V.1
- Tatiana Tkatch1
- 1Northwestern University
Protocol Citation: Tatiana Tkatch 2023. Quantitative real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwd4o2lmk/v1Version created by Christine Weber-Schmidt
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: December 18, 2023
Last Modified: December 18, 2023
Protocol Integer ID: 92468
Keywords: qPCR, real time PCR
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-020551
NIH
Grant ID: NS 34696
JPB Foundation
CHDI Foundation
Abstract
Quantitative real-time PCR protocol used for Day et al.
Materials
-SuperScript IV VILO Master Mix (Thermo Fisher Scientific)
-TaqMan PreAmp Master Mix (Thermo Fisher Scientific)
-TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific)
-UltraPure DNase/RNase-Free Distilled Water (Thermo Fisher Scientific)
-TaqMan Gene Expression Assays: hprt Mm03024075_m1, Slc12a2(NKCC1) Mm01265955_m1,Slc12a5(KCC2)
Mm00803929_m1, Slc4a3 Mm00436654_g1, Slc4a10 Mm00473827_m1 (Thermo Fisher Scientific)
-PCR tubes, PCR plates and film
-Micropipettes
-Aerosol-resistant barrier pipette tips
gDNA digestion
gDNA digestion
51m 15s
-For each sample prepare 10ul gDNA digestion reaction mix according to SuperScript IV VILO Master Mix protocol (see pdf attached or substeps below).
Digest gDNA for 00:02:00 min at 37 °C .
2m
Place the tubes on ice.
Add SuperScript IV VILO Master Mix and Nuclease-free water.
Gently mix and incubate at 25 °C for 00:10:00 .
10m
Then at 50 °C for 00:10:00 .
10m
Inactivate enzyme by incubation at 85 °C for 00:05:00 .
5m
Pre-amplification
Pre-amplification
51m 15s
Perform Pre-amplification according to TaqMan PreAmp Master Mix protocol (see pdf attached or substeps below).
-TaqMan PreAmp Master Mix 25ul, pooled assay mix (0.2X) 12.5ul, cDNA 2ul, nuclease free water, total 50ul.
Run reaction settings: 95 °C for 00:10:00 then 95 °C for 00:00:15 , 60 °C for 00:04:00 (10 cycles), inactivate enzyme 99 °C for 00:10:00 , hold at 4 °C
24m 15s
-Dilute each reaction 10 times.
Amplification
Amplification
2m 41s
-PCR reaction mix: Gene Expression Assay (20X) 1ul, Preamplified cDNA product 5ul, TaqMan Fast Advanced Master Mix (2X) 10ul, nuclease free water 4ul, total 20ul.
-Run the reactions: Using incubation 50 °C for 00:02:00 then enzyme activation-95 °C for 00:00:20 , Denature 95 °C for 00:00:01 , Anneal/Extend 60 °C for 00:00:20 40 cycles.
2m 41s
Analysis
Analysis
Experimental Ct values were normalized to hprt values using the following
formula: ΔCt = Ct (gene of interest) − Ct (hprt). The final expression levels were shown as ΔCt
values.