Cells (2.0 × 105) were seeded in six-well plates and then IGF2BP2 shRNA or RUNX2 siRNAs were transfected into cells. RNA-Quick Purification Kit (Vazyme, Nanjing, China) was used for total RNA isolation from cells. 1 µg total RNA was converted to cDNA for RT-qPCR using HiScript II Q RT SuperMix for RT-qPCR (Vazyme, Nanjing, China). An Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems) using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China) was used for real-time quantitative RT-qPCR analysis. To standardize the input cDNA, β-actin was run in parallel as reference. The primer sequences are provided in Supplementary Table S1 .
Hiscript 2 q rt supermix for rt qpcr
Manufactured by Vazyme
Sourced in China
HiScript II Q RT SuperMix for RT-qPCR is a one-step reverse transcription and real-time PCR reagent. It contains a high-performance reverse transcriptase and a hot-start DNA polymerase, optimized for sensitive and specific quantification of RNA targets.
Automatically generated - may contain errors
Lab products found in correlation
Rna quick purification kit, by Vazyme (1 mentions)
Applied biosystems 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Aceq qpcr sybr green master mix, by Vazyme (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time system, by Bio-Rad (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Chamq sybr color qpcr master mix, by Vazyme (1 mentions)
Rnaprep pure micro kit, by Tiangen Biotech (1 mentions)
M5 hiper real time pcr super mix with low rox, by Mei5 Biotechnology (1 mentions)
Lightcycler 480 instrument 2, by Roche (1 mentions)
4 protocols using hiscript 2 q rt supermix for rt qpcr
1
Gene Expression Analysis by RT-qPCR
2
Quantitative RT-PCR Protocol for Gene Expression
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Total RNA was extracted using TRIzol reagent (Invitrogen, 15596026, USA). Total cDNA was prepared with HiScript II Q RT SuperMix for RT-qPCR (Vazyme, R223-01, China). qRT-PCR was then performed using specific primers in a CFX96 Real-Time System (Bio-Rad, USA). Primers are listed in Supplementary Table 2 .
3
Gene Expression Analysis of Orbital Tissues
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Total RNA of orbital connective tissues and OFs was extracted using an RNA-Quick Purification Kit (Yishan Biotechnology, Shanghai, China). The cDNA was synthesized using a HiScript II Q RT SuperMix for RT-qPCR (Vazyme, Nanjing, China). RT-qPCR was performed on a Roche LightCycler 480 (Roche, Basel, Switzerland) with the ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China). GAPDH was used for normalization of results. Table 2 displayed the gene-specific primer sequences for RT-qPCR.
4
Single-cell and Bulk-cell RT-qPCR Protocols
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For single-cell RT-qPCR, cDNA from individual cells was diluted with water in a ratio of 1:20. RT-qPCR was performed using 2× M5 HiPer Realtime PCR Super mix with Low Rox (Mei5 Biotechnology, MF797) on a Roche LightCycler® 480 Instrument II. For bulk-cell RT-qPCR, total RNA was extracted from the collected cells using the RNAprep Pure Micro kit (Tiangen, DP420), and reverse transcription was performed with HiScript II Q RT SuperMix for RT-qPCR (Vazyme, R223-1). Primer sequences are listed in Supplementary information, Table S9 .
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