Total RNA from normal human renal tissues and cultured HK-2 cells was isolated with TriReagent (Sigma). qRT-PCR analysis was carried out using SYBR Green PCR master mix (Applied Biosystems) on Bio-Rad iQ5 Multicolor Real-Time qRT-PCR Detection system (Bio-Rad, Hercules, CA, USA), in one-step RT-PCR protocol as previously described [21 (link)]. The relative expressions of IL-6, TNF-α and IκBα mRNA were normalized to GAPDH expression and examined by using the 2−ΔΔCT method [22 (link)]. The primers used in this study were listed as follows: IL-6, forward 5′-ATGAACTCCTTCTCCACAAGCGC-3′, reverse 5′-GAAGAGCCCTCAGGCTGG ACTG-3′; TNF-α, forward 5′-CAGGGGCCACCACGCTCTTC-3′, reverse 5′-CTTGGGGCAGGGGCTCTTGAC-3′; IκBα, forward 5′-CGTGTCTGCACCTAGCCTCTATC-3′, 5′-GCGAAACCAGGTCAGGATTC-3′.
Iq5 multicolor real time qrt pcr detection system
Manufactured by Bio-Rad
Sourced in United States
The Bio-Rad iQ5 Multicolor Real-Time qRT-PCR Detection system is a laboratory equipment designed for real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis. It features a multicolor detection system and is capable of performing real-time monitoring of multiple gene targets simultaneously.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop spectrophotometer, by Thermo Fisher Scientific (4 mentions)
Sybr green pcr master mix, by Takara Bio (2 mentions)
Reverse transcription system kit, by Thermo Fisher Scientific (2 mentions)
Taqman microrna assay kit, by Thermo Fisher Scientific (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Dna eraser, by Takara Bio (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
5 protocols using iq5 multicolor real time qrt pcr detection system
1
Quantifying Inflammatory Gene Expression
2
Quantifying gene and miRNA expression in cell lines
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The RNA in the two cell lines was extracted with TRIzol reagent (Invitrogen) following the instruction of manufacturer. The concentration and purity of extracted RNA were detected by a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Then cDNA was synthesized using the Reverse Transcription System Kit (Applied Biosystems, Foster City, CA, USA). The detection of ZFAS1, GAPDH and miR-100-3p expression was performed by RT-PCR using SYBR Green PCR Master Mix (Takara, Dalian, China) and TaqMan MicroRNA Assay Kit (Applied Biosystems) on Bio-Rad iQ5 Multicolor Real-Time qRT-PCR Detection system (Bio-Rad, Hercules, CA, USA). GAPDH was a reference gene for ZFAS1 and METTL3, while U6 was the reference gene for miR-100-3p. The primers were synthesized by Sangon (Additional file 5 : Table S2) (Shanghai, China). All statistics were analyzed based on 2−ΔΔCt method.
3
Quantification of MCM6, GAPDH, and miR-23b-3p
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The RNA in the two cell lines was extracted with TRIzol reagent (Invitrogen) following the instruction of manufacturer. The concentration and purity of extracted RNA were detected by a NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Then cDNA was synthesized using the Reverse Transcription System Kit (Applied Biosystems, Foster City, CA, USA). The detection of MCM6, GAPDH and miR-23b-3p expression was performed by RT-PCR using SYBR Green PCR Master Mix (Takara, Dalian, China) and TaqMan MicroRNA Assay Kit (Applied Biosystems) on Bio-Rad iQ5 Multicolor Real-Time qRT-PCR Detection system (Bio-Rad, Hercules, CA, USA). GAPDH was a reference gene for SNHG16 and MCM6, while U6 was the reference gene for miR-23b-3p. The primers for SNHG16, MCM6, GAPDH, miR-23b-3p and U6 (Table 1) were synthesized by Guangzhou Ribo-Bio (Guangzhou, China). All statistics were analyzed based on 2 -ΔΔCt method.
4
Quantitative analysis of long non-coding RNAs
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Briefly, cDNA was synthesized from total RNA using a PrimeScript RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). Primers for lncRNAs were designed and synthesized. Then, qPCR assays were performed using a Bio-Rad iQ5 Multicolor Real-Time qRT-PCR Detection system (Bio-Rad, Hercules, CA, USA). The expression levels of lncRNAs were detected as previously described (21 (link)). Human β-actin was used as a housekeeping gene for normalization. The expression levels of lncRNAs were measured in terms of the cycle threshold (CT) and were then normalized to β-actin expression using the 2−ΔΔCt method (21 (link)).
5
Quantifying Cancer Stem Cell Markers
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Briefly, cDNA was synthesized from total RNA using a PrimeScript RT reagent kit with a DNA Eraser (Takara, Shiga, Japan). Primers for genes related to cancer stem cells were designed and synthesized. Then, qPCR assays were performed using a Bio-Rad IQ5™ Multicolor Real-Time qRT-PCR detection system (Bio-Rad, Hercules, CA, USA). The mRNA expression levels were detected as previously described (5 (link)) using specific primer sequences (Table I ). The expression levels were measured in terms of the cycle threshold (Ct) and were then normalized to GAPDH expression using the 2−ΔΔCt method (5 (link),14 (link)).
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