Total cellular RNAs were extracted from A549 cells. Stem-loop RT-PCR primers for human miR-10a, miR-205, miR-221, miR-222, and U6 were synthesized by Ribobio (Guangzhou, China). For miR-10a, miR-205, miR-221, miR-222, and U6 detection, RNA samples were reverse-transcribed into cDNA using Revert Ace transcriptase by specific stem-loop RT primers according to the manufacturer's instruction. Transcript levels were measured against an endogenous control by quantitative PCR using the SYBR Green I fluorogenic dye using the Mastercycler ep realplex system (Eppendorf, Hamburg, Germany).
Stem loop rt pcr primers
Manufactured by RiboBio
Sourced in China
Stem-loop RT-PCR primers are designed for the detection and quantification of specific RNA targets, including microRNAs, through reverse transcription and real-time PCR amplification. These primers form a stem-loop structure that enhances the specificity and sensitivity of the RT-PCR reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Revert ace kit, by Toyobo (2 mentions)
Rnase free dnase, by Thermo Fisher Scientific (2 mentions)
Primescript rt master mix, by Takara Bio (2 mentions)
Mastercycler ep realplex system, by Eppendorf (1 mentions)
Sybr green qrt pcr, by RiboBio (1 mentions)
5 protocols using stem loop rt pcr primers
1
Quantification of miRNA Transcripts in A549 Cells
2
Quantifying HOTTIP and HOXA13 gene expression
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After isolation from culture cells or clinical tissues with Trizol reagent (Invitrogen), each RNA sample was treated with RNase-Free DNase to remove genomic DNA (Invitrogen). These RNA samples were then reverse transcribed into cDNAs using Revert Ace kit (TOYOBO, Osaka, Japan). Human miRNAs and U6 were detected with their specific stem-loop RT-PCR primers (Ribobio, Guangzhou, China) [53 (link)]. HOTTIP, HOXA13, and other potential HOTTIP downstream gene expression were measured through the SYBR-Green qRT-PCR. The expression of individual gene was calculated relative to the β-actin or GAPDH expression [53 (link)–55 (link)].
3
Gene Expression Analysis by RT-qPCR
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Total RNA was isolated from culture cells or tissue specimens with Trizol reagent (Invitrogen, 94402). To remove genomic DNA, each RNA sample was treated with DNase I (RNase-free) (Thermo Fisher,18068015). Each RNA sample was then reverse transcribed into cDNAs using PrimeScriptTM RT Master Mix (TaKaRa, RR036A). Human miRNAs and U6 were detected with their specific stem-loop RT-PCR primers (Ribobio, Guangzhou, China) 47 (link),48 (link). The mRNA expression of DAZAP1 and other genes were examined with their specific RT-qPCR primers (Table S1 ).
4
RNA Extraction, Reverse Transcription, and qPCR Analysis
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Total RNA was isolated from culture cells or tissue specimens with Trizol reagent (Invitrogen, 94402). To remove genomic DNA, each RNA sample was treated with DNase I (RNase-free) (Thermo Fisher, 18068015). Each RNA sample was then reverse transcribed into cDNAs using PrimeScriptTM RT Master Mix (TaKaRa, RR036A). Human miRNAs and U6 were detected with their specific stem-loop RT-PCR primers (Ribobio, Guangzhou, China) using a Quantitative PCR instrument (QuantStudio™ 6, ABI, USA) 30 (link). The relative mRNA expression of MED13L, PRKCA and other genes were calculated by using the 2-ΔΔCt method. Indicated primers are listed in Supplementary Table 1 . Each sample was examined at least in triplicate. PCR product specificity was confirmed by a melting-curve analysis.
5
Reliable RNA Isolation and miRNA Quantification
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Total RNA was isolated from H1299 and PC9 cells with Trizol reagent (Invitrogen). To avoid contamination of genomic DNA, each RNA sample was treated with RNase-Free DNase to remove genomic DNA (Invitrogen). The corresponding cDNAs were generated using the Revert Ace kit (TOYOBO, Osaka, Japan). Human miR-608, miR-4513, and U6 small RNA were examined with their specific stemloop RT-PCR primers (Ribobio, Guangzhou, China) using SYBR-Green qRT-PCR as described previously [28, 29] .
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