These procedures were performed as described previously63 (link), 64 (link). Primers used in RT-qPCR are indicated in Supplementary Table 1 . The miR-122, miR-16 (as internal control for vesicular miR-122), and U6 primers (as internal control for intracellular miR-122) were purchased from Qiagen. Determination of PKM isoforms was performed as described65 (link). Products of RT-PCR were digested with NcoI, PstI, or both enzymes, plus an uncut control, and separated on an agarose gel with Sybr safe. The presence of a PstI digestion site indicates the splicing isoform M2 whereas the NcoI site indicates isoform M1.
Mir 16
Manufactured by Qiagen
Sourced in United States
The MiR-16 is a laboratory instrument designed for the quantitative analysis of microRNA (miRNA) expression. It utilizes real-time PCR technology to accurately measure the levels of specific miRNA molecules in biological samples. The core function of the MiR-16 is to provide researchers with a reliable tool for the detection and quantification of miRNA targets.
Automatically generated - may contain errors
Lab products found in correlation
U6 primer, by Qiagen (2 mentions)
Mir 122, by Qiagen (2 mentions)
Interleukin 6 (il 6), by Qiagen (1 mentions)
Ifn β, by Qiagen (1 mentions)
Ifn γ, by Qiagen (1 mentions)
Tnf α, by Qiagen (1 mentions)
Microm hm550, by Thermo Fisher Scientific (1 mentions)
Snord95, by Qiagen (1 mentions)
Sybr green master mix kit, by Qiagen (1 mentions)
4 protocols using mir 16
1
Quantifying Cellular miRNA and Isoform Expression
2
Quantifying Cellular miRNA and Isoform Expression
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These procedures were performed as described previously63 (link), 64 (link). Primers used in RT-qPCR are indicated in Supplementary Table 1 . The miR-122, miR-16 (as internal control for vesicular miR-122), and U6 primers (as internal control for intracellular miR-122) were purchased from Qiagen. Determination of PKM isoforms was performed as described65 (link). Products of RT-PCR were digested with NcoI, PstI, or both enzymes, plus an uncut control, and separated on an agarose gel with Sybr safe. The presence of a PstI digestion site indicates the splicing isoform M2 whereas the NcoI site indicates isoform M1.
3
Quantifying Inflammatory Gene Expression
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A total of 80 μM of tissue was macrodissected using a cryostat (Fisher Scientific, Waltham, MA, USA; Microm HM 550). Briefly, RNA was isolated, reverse transcribed, and RT-PCR was performed using rat primers; CCL2 (Qiagen, Valencia, CA, USA, # PPR06714B), IFNβ (Qiagen, Valencia, CA, USA, #PPR06442B), IL6 (Qiagen, Valencia, CA, USA, # PPR06483B), IFNγ (Qiagen, Valencia, CA, USA, #PPR45050C), and TNFα (Qiagen, Valencia, CA, USA, #PPR06411F) [37 (link)]. Raw data were exported from the real-time instrument software and relative gene expression (RQ) was calculated using the ΔΔ-Ct method. Specific RT2 Primer Assays Endogenous controls run on each plate included SNORD-95 (Qiagen, Valencia, CA, USA, #331452), and miR16 (Qiagen, Valencia, CA, USA, #331452). All samples were normalized against pathologically confirmed normal esophagus and were run in technical duplicates.
4
Quantifying miR-146a and miR-155 Expression
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miR146a and miR-155 were detected by Real-time PCR assays by using the SYBR Green Master Mix kit (QIAGEN, MD, USA). miRNA primers were purchased from QIAGEN (has-miR-146A-5p, Cat. No YP00204688; has-miR-155-5p, Cat no. YP00204308), and quantitative PCR was performed using Real-time PCR (Roche, Manheim, Germany). The Real-time PCR program included the following steps: an initial denaturation step at 95°C for 10 min; 45 amplification cycles that consisted of a denaturation step (10 s at 95°C) and an annealing step (60 s at 60°C). Expression levels of miRNAs were normalized to the level of miR-16 (QIAGEN, MD, USA) as a control miRNA using the 2– ΔΔCt method.
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