Total RNA was isolated by using Quick-RNA miniPrep kit (Zymo Research, Irvine, CA) from cell lines and murine and human liver tissues, HCC, CCA, and adjacent non-tumorous tissues. Gene expression was assessed using real-time PCR. Total RNA was subjected to reverse transcription (RT) by using M-MLV Reverse transcriptase (Lucigen, Middleton, WI). TaqMan probes for human and murine PHB1, MAT1A, c-MYC, MAFG, c-MAF, glutamate-cysteine ligase catalytic and modifier subunits (Gclc and Gclm), and the Universal PCR Master Mix were purchased from Bio-Rad (Hercules, CA). Hypoxanthine phosphoribosyltransferase 1 or glyceraldehyde 3-phosphate dehydrogenase (GAPDH, for human CCA) was used as a housekeeping gene. The thermal profile consisted of an initial denaturation at 95°C for 3 minutes followed by 40 cycles at 95°C for 3 seconds and at 60°C for 30 seconds. The cycle threshold (Ct value) of the target genes was normalized to that of the housekeeping gene to obtain the delta Ct (ΔCt). The ΔCt was used to find the relative expression of target genes according to the formula: relative expression= 2-ΔΔCt, where ΔΔCt= ΔCt of target genes in experimental condition – ΔCt of target gene under control condition.
Universal pcr master mix
Manufactured by Bio-Rad
Sourced in United States
Universal PCR Master Mix is a ready-to-use solution for performing polymerase chain reaction (PCR) experiments. It contains all the necessary components, including DNA polymerase, dNTPs, and reaction buffer, to efficiently amplify DNA sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Quick rna miniprep kit, by Zymo Research (1 mentions)
High capacity cdna kit, by Thermo Fisher Scientific (1 mentions)
Cfx connect real time system cycler, by Bio-Rad (1 mentions)
Mir 145, by Thermo Fisher Scientific (1 mentions)
Mir 125a 5p, by Thermo Fisher Scientific (1 mentions)
Taqman assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using universal pcr master mix
1
Quantifying Gene Expression in Liver Diseases
2
Quantitative Real-Time PCR for Fusobacterium
3
Quantitative Real-Time PCR Analysis
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Total RNA was isolated with Trizol (Invitrogen). Total RNA (1–2 µg) was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Invitrogen). Two microliters of RT product were subjected to quantitative real-time PCR analysis. TaqMan probes for human MAT1A (Hs01547962_m1), PIN1 (Hs01598308), and CSNK2A1, and murine Mat1a (Mm00522563_m1), Pin1 (Mm03053328_s1), and Csnk2a1 (Mm00786779_s1) were purchased from Applied Biosystems. Universal PCR Master Mix was purchased from Bio-Rad. Ubiquitin C was used as housekeeping gene (Hs01871556_s1 and Mm02525934_g1). The cycle Ct value of the target genes was normalized to that of the housekeeping gene to obtain the delta Ct (∆Ct). The ΔCt obtained was used to find the relative expression of target genes according to the formula: relative expression = 2 − ΔΔCt, where ΔΔCt = ΔCt of target genes in experimental groups −ΔCt of target genes in control group.
4
Quantitative PCR for Gene Expression
5
Quantification of miRNA and mRNA Expression
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miRNA qRTPCR analysis was performed using Taqman assays (Applied Biosystems) (Cat #4429795): miR-145 (Assay #2278), miR-125a-5p (Assay #2198). The endogenous control was U6 snRNA (Assay #1973). Analysis of mRNA expression for Ret and DAT used (Cat #4331182, Rn00562224_m1) and (Cat #4331182, Rn01463098_m1) respectively. GAPDH (Cat #4331182, Rn01775763_m1) was selected as an endogenous control [56 (link)]. Analyses were performed with a final volume of 10 µL of miRNA cDNA, or 20 µL of mRNA cDNA and Universal PCR Mastermix (#4369016) in a Bio-Rad CFX Connect Real-time system cycler (Bio-Rad, CA, USA). Each sample was run in triplicate. Expression was normalized (∆Ct) using the appropriate endogenous control; small nuclear RNA U6 for the miRNA, and GAPDH for the mRNA analyses. A one-tailed T-test (comparison of means) was used to test for significance, in line with the differential expression observed for the array data.
6
Quantitative gene expression analysis
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Total RNA was isolated with Trizol (Invitrogen). Total RNA (1-2 µg) was reverse transcribed into cDNA using M-MLV Reverse Transcriptase (Invitrogen). Two microliters of RT product were subjected to quantitative real-time PCR analysis. TaqMan probes for human MAT1A (Hs01547962_m1), DNAJC15 (Hs00387763_m1), and murine Mat1a (Mm00522563_m1), Dnajc15 (Mm00481271_m1) and Phb1 (Mm01627033) were purchased from Applied Biosystems. Universal PCR Master Mix was purchased from Bio-Rad. Ubiquitin C was used as housekeeping gene (Hs01871556_s1 and Mm02525934_g1). The cycle Ct value of the target genes was normalized to that of the housekeeping gene to obtain the delta Ct (∆Ct). The ΔCt obtained was used to find the relative expression of target genes according to the formula: relative expression = 2-ΔΔCt, where ΔΔCt = ΔCt of target genes in experimental groups - ΔCt of target genes in control group.
7
Quantifying Fusobacterium in Ulcerative Colitis
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Quantitative real-time PCR analysis for Fusobacterium was performed using the Universal PCR Master Mix (Bio-Rad) and StepOnePlus™ Real-Time PCR System (Applied Biosystems). The pan-fusobacterium TaqMan primer/probe set used in this study were described previously [13 (link), 39 (link)]. The cycle threshold (Ct) values for pan-fusobacterium were normalized to the amount of human DNA in each reaction by using a primer/probe set for the reference gene, prostaglandin transporter (PGT), as described previously [40 (link)]. All assays were done in duplicate and we averaged the results. We have reported that subset of UC cases show heavy enrichment of Fusobacterium in the inflamed colonic mucosa [22 (link)]. In this cohort, we identified ten cases (11.6%) of UC with enrichment of Fusobacterium using the same cut off value [22 (link)]. We then defined these cases as Fusobacterium high (FB-high) cases. Since the amount of Fusobacterium in detectable cases except the FB-high cases was much lower than that of FB-high cases and had no relevance to the clinic-pathological features of patients [22 (link)], we then attached these cases with Fusobacterium undetectable cases and defined as Fusobacterium low and negative (FB-low/neg) cases.
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