The cDNA synthesis for qPCR was transcribed through a OneScript cDNA Synthesis Kit (ABM, Richmond, ON, Canada). The levels of mRNA were quantified through quantitative real-time PCR (qRT-PCR) in StepOne Plus (Applied Biosystems), using KicqStart Kit (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) with specific primers (sequences in Table S5 ). All the procedures were conducted according to the manufacturer’s protocol. The relative expressions were calculated using the 2−ΔΔCt method [25 (link)]. The control transcripts used for 2−ΔΔCt were ATP5F1 and RPLP2 as a housekeeper for the mRNA [16 (link),23 (link)].
Kicqstart kit
Manufactured by Merck Group
Sourced in United States
The KicqStart Kit is a laboratory equipment product offered by Merck Group. It is designed for basic laboratory procedures. The kit includes essential components required for various laboratory applications. The core function of the KicqStart Kit is to provide a comprehensive set of tools for general laboratory work.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Onescript cdna synthesis kit, by Applied Biological Materials (2 mentions)
Steponeplus, by Thermo Fisher Scientific (2 mentions)
Stepone plus equipment, by Thermo Fisher Scientific (1 mentions)
Cdna synthesis kit, by Applied Biological Materials (1 mentions)
3 protocols using kicqstart kit
1
cDNA Synthesis and qRT-PCR Analysis
2
Quantification of Calcium Channel Gene Expression
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Frozen lung samples (∼100 mg) were homogenized in TRIzol (Thermo Fisher, Waltham, MA,
USA). RNA was resuspended in nuclease-free water and the absorbance was measured at 260
and 280 nm. The 260/280 absorbance ratio was 1.9–2.05. The RNA was stored at −80°C until
use. The cDNA synthesis was transcribed through OneScript cDNA Synthesis Kit (ABM,
Richmond, Canada). Gene expression of TRPC1, TRPC4, TRPC6, ORAI1, ORAI2, and 18S was
quantified through quantitative realtime PCR (qRTPCR) in StepOne Plus (Applied Biosystems,
Foster City, CA, USA), using KicqStart Kit (Sigma-Aldrich; seeTable 1 ). All procedures were realized according to
the manufacturer’s protocol. The relative expressions were calculated using the 2−ΔΔCt
method. The control group was used as reference and 18S gene as housekeeper.![]()
USA). RNA was resuspended in nuclease-free water and the absorbance was measured at 260
and 280 nm. The 260/280 absorbance ratio was 1.9–2.05. The RNA was stored at −80°C until
use. The cDNA synthesis was transcribed through OneScript cDNA Synthesis Kit (ABM,
Richmond, Canada). Gene expression of TRPC1, TRPC4, TRPC6, ORAI1, ORAI2, and 18S was
quantified through quantitative realtime PCR (qRTPCR) in StepOne Plus (Applied Biosystems,
Foster City, CA, USA), using KicqStart Kit (Sigma-Aldrich; see
the manufacturer’s protocol. The relative expressions were calculated using the 2−ΔΔCt
method. The control group was used as reference and 18S gene as housekeeper.
Primers sequence’s characteristics uses in the analysis.
| Gene | F/R | Primer sequence (5′–3′) | Tm (°C) | Product | Extension |
|---|---|---|---|---|---|
| length (bp) | Time (s) | ||||
| TRPC1 | For | 5′-AGTTCCTGAACACCGTTTGG-3′ | 60° | 164 bp | 10 s |
| Rev | 5′-CGGTGTGTGAATGATTCTGC-3′ | ||||
| TRPC4 | For | 5′-CAGGCTGGAGGAGAAGACAC-3′ | 60° | 195 bp | 10 s |
| Rev | 5′-AGGCTAGCAGCAGCAGAAAC-3′ | ||||
| TRPC6 | For | 5′-TCTGGCTGCTCATTGCCAGGAATA-3′ | 60° | 323 bp | 30 s |
| Rev | 5′-AGAGTGGCTGAAGGAGTCATGCTT-3′ | ||||
| ORAI1 | For | 5′-CGCCCTCATGATCAGTACCT-3′ | 60° | 191 bp | 10 s |
| Rev | 5′-AGAACTTCACCCAGCAGAGC-3′ | ||||
| ORAI2 | For | 5′-GTGGGTCTCATCTTCGTGGT-3′ | 62° | 142 bp | 10 s |
| Rev | 5′-CCACCTGTAGGCTTCTCTCG-3′ | ||||
| 18S | For | 5′-GTAACCCGTTGAACCCCATT-3′ | 58° | 152 bp | 10 s |
| Rev | 5′-CCATCCAATCGGTAGTAGCG-3′ |
3
Quantifying Gene Expression in Lung Samples
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Frozen left lung samples (∼100 mg) were homogenized in TRIzol (Thermo Fisher Scientific, Waltham, MA, United States). The RNA was resuspended in nuclease-free water (20 μl) to later determination of its concentration and purity by spectrophotometry, being the 260/280 ratio of all samples in the range of 1.9–2.0. The RNA was stored at −80°C until later use. Synthesis to cDNA was performed using the cDNA Synthesis Kit (ABM, Richmond, Canada). The gene expression of TRPC1, TRPC4, TRPC6, ORAI1, ORAI2 and 18S was determined by real-time PCR (qPCR) in a StepOne Plus equipment (Applied Biosystems, Foster City, CA, United States), using a KicqStart kit (Sigma-Aldrich, United States) with the primers sequence used by Castillo-Galán et al. (2020) (link). Relative gene expression was calculated with the 2–ΔΔCt method using 18S gene.
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