7500 fast system realtime pcr cycler

Total RNA from intestinal fibroblasts was extracted using the miRNeasy kit (Qiagen) according to the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop Spectrophotometer (NanoDrop Technologies, USA) and 1 μl was run on an agarose gel (1%) to assess RNA quality. RNA samples were reverse transcribed using a High-Capacity-RNA to cDNA kit (Applied Biosystems, USA) in a 20-μl reaction. cDNA was then incubated with TaqMan assays (MCL-1L/MCL-1ES, MCL-1S, IL6, IL8, COL1A2, COL3A1 or GAPDH) and TaqMan Universal MasterMix (Applied Biosystems) on a 7500 Fast System RealTime PCR cycler (Applied Biosystems) according to the manufacturer’s instructions. The Taqman probe for MCL-1L also detects the MCL-1ES isoform while there is no commercially available probe for just MCL-1ES. A separate probe for selective MCL-1S was used. Fold-changes were calculated using the 2^-ΔΔCt method normalised to GAPDH.

Total RNA was extracted using TRIzol Reagent (ThermoFisher) according to the manufacturer’s instructions. cDNA synthesis was performed using High Capacity cDNA Reverse Transcriptase kit (ThermoFisher). qPCR reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems) on a 7500 Fast System RealTime PCR cycler (Applied Biosystems). Primer sequences used in this study are:
F9 Forward5′-CAGTGTTCAGAGCCAAGCAA-3′
F9 Reverse5′-CATGGTGAACACGAAACCAG-3′
PROZ Forward5′-CACCCCTGAGAAAGACTTCG-3′
PROZ Reverse5′-GGAGCCTCTGTGTTCTCTGG-3′
RB Forward5′-AACCCAGGAAGGAATGGCT-3′
RB Reverse5′-CTGCGTTCAGGTGATTGATG-3′
IL8 Forward5′-GAGTGGACCACACTGCGCCA-3′
IL8 Reverse5′-TCCACAACCCTCTGCACCCAGT-3′
IL6 Forward5′’-CCAGGAGCCCAGCTATGAAC-3′
IL6 Reverse5′-CCCAGGGAGAAGGCAACTG-3′
CCL20 Forward5′-GGCGAATCAGAAGCAGCAAGCAAC-3′
CCL20 Reverse5′-ATTGGCCAGCTGCCGTGTGAA-3′
IL1A Forward5′-AGTGCTGCTGAAGGAGATGCCTGA-3′
IL1A Reverse5′-CCCCTGCCAAGCACACCCAGTA-3′
IL1B Forward5′-TGCACGCTCCGGGACTCACA-3′
IL1B Reverse5′-CATGGAGAACACCACTTGTTGCTCC-3′
CDKN1A Forward5′-CCTGTCACTGTCTTGTACCCT-3′
CDKN1A Reverse5′-GCGTTTGGAGTGGTAGAAATCT-3′
ACTIN Forward5′-GCCCTGAGGCACTCTTCCA-3′
ACTIN Reverse5′-CGGATGTCCACGTCACACTTC-3′
RSP14 Forward5′-CTGCGAGTGCTGTCAGAGG-3′
RSP14 Reverse5′-TCACCGCCCTACACATCAAACT-3′

Blood miRNAs were isolated by PAXgene Blood miRNA Kit. Primers specific for miR-34a were obtained from Applied Biosystems (Foster City, CA, USA) to execute quantitative real-time PCR according to the vendor's protocol for TaqMan miRNA assays. Isolated total RNA from human plasma was initially processed for reverse transcription (RT) using two miRNA-specific primers (miR-34a: AB Assay ID 000426) to obtain RT products that were subsequently used for quantitative real-time PCR on a 7500 Fast System Real-Time PCR cycler (Applied Biosystems). U6 was used as a control to calculate the relative expression levels of miR-34a. Numerical indices of these expression levels are expressed using the 1/ΔCT method, and the values for each individual microRNA were obtained after subtracting the CT value of snoRNA202 (AB assay ID 001232, Applied Biosystems.

Cells were washed with PBS and lysed directly into the culture dish using TRIzol Reagent (Thermo Fisher). cDNA synthesis was performed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher). qPCR reactions were performed using SYBR Green PCR Master Mix (Applied Biosystems,) on a 7500 Fast System RealTime PCR cycler (Applied Biosystems). Primer sequences are listed in Table S2.

Quantitative real‐time PCR was carried out using SYBR Green I (Sigma‐Aldrich) assay reagents to verify gene expression profiles. Two hundred nanograms of total RNA were reverse transcribed to cDNA; real‐time PCR reactions were performed on a 7500 Fast System Real‐Time PCR cycler (Applied Biosystems, Foster City, CA), according to the manufacturer's instructions. Changes in gene expression between different treatment groups were calculated using the delta Ct method.
Primer sequences:

tyra−3	Forward Primer	AGCACGACTGCCACAATTA
	Reverse Primer	GTGGAGAGTTCGAGCCTAATG
ins−35	Forward Primer	ACGCGCACTAAAGGTCTATTC
	Reverse Primer	GTTGAGCCACATCCTTCCAT
act−1	Forward Primer	CAACACTGTTCTTTCCGGAG
	Reverse Primer	CTTGATCTTCATGGTTGATGGG