The experiments were done using transcript target‐specific primer pairs (Table A2 in Appendix 1 ) and the QuantiNova® SYBR® Green RT‐PCR Kit as recommended by the manufacturer (Qiagen, LLC ). All primer pairs were optimized to have >90% amplification efficiency (as judged by standard curves generated from genomic DNA) and to produce a unique amplicon (as judged by melt curve analysis) of the expected size (as judged by agarose gel electrophoresis). RNA samples used for RT‐qPCR were DNase‐treated and verified to be free of DNA contamination by PCR. One‐step RT‐qPCR reactions (10 µl, 30 pg of RNA template) were run in a Rotor‐Gene Q light cycler (Qiagen, LLC). Two‐step PCR cycling was performed as follows: 95°C for 5 s and 60°C for 10 s for 40 cycles. For each transcript target, the threshold cycle (Ct) was normalized to the Ct of the standard mycobacterial internal calibrator sigA transcript to generate transcript relative abundance data as reported (Dubnau, Fontan, Manganelli, Soares‐Appel, & Smith, 2002 ; Livak & Schmittgen, 2001 ).
Rotor gene q lightcycler
Manufactured by Qiagen
The Rotor-Gene Q is a real-time PCR cycler designed for nucleic acid analysis. It features a unique rotor-disc format and uses fluorescence detection to quantify and analyze DNA, RNA, and other target molecules.
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Lab products found in correlation
Sensifast sybr no rox one step kit, by Meridian Bioscience (4 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (3 mentions)
Quantinova sybr green rt pcr kit, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Sensifast no rox kit, by Meridian Bioscience (1 mentions)
Sensimix sybr kit, by Meridian Bioscience (1 mentions)
Superscript 3, by Thermo Fisher Scientific (1 mentions)
Rna bee, by Tel-Test (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Gotaq dna polymerase, by Promega (1 mentions)
Oligo dt primer, by Thermo Fisher Scientific (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Qiazol, by Qiagen (1 mentions)
Rotor gene sybr green, by Qiagen (1 mentions)
Nucleospin rna extraction kit, by Macherey-Nagel (1 mentions)
Qscript cdna supermix, by Quanta Biosciences (1 mentions)
10 protocols using rotor gene q lightcycler
1
Optimized RT-qPCR for Mycobacterial Gene Expression
2
Quantitative Analysis of Gene Expression
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Gene expression analysis by quantitative real-time PCR was performed for every strain with at least four biological replicates and two technical replicates. Bacteria were grown until an OD600 of 0.9 to 1.2 with or without addition of EDTA was reached after 1.5 h of growth, and total RNA was isolated using an RNeasy kit (Qiagen). DNA was removed by DNase treatment using a Turbo DNA-free kit (Ambion). Reverse transcription and quantitative real-time PCR were performed using a SensiFast SYBR No-ROX One-Step kit (Bioline) in a Rotor-Gene Q LightCycler (Qiagen). Analysis of the relative mRNA level changes was done according to the method described by Pfaffl (51 (link)). MRNA levels were normalized to the mRNA levels of the gmk, rpoD, and gyrB reference genes as described before (52 (link)). For statistical analysis, an unpaired t test was performed using GraphPad Prism version 6.0 software.
3
Quantitative RT-PCR Analysis of Gene Expression
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Strains were grown under static growth conditions in LB medium and total RNA isolation was performed using the RNeasy minikit (Qiagen). For removal of genomic DNA, RNA was treated with DNase using the TURBO DNA-free kit (ambion). Reverse transcription and quantitative real-time PCRs (qRT-PCR) were performed using the SensiFast SYBR No-ROX One Step kit (Bioline) in a Rotor-Gene Q lightcycler (Qiagen). Relative changes in mRNA levels were analyzed according to Pfaffl [60 (link)] and normalized against the transcription levels of multiple reference genes according to the method described by Vandesompele et al. [61 ]. The reference genes gyrB, gmk and rpoD were used as previously described [22 (link), 59 (link)].
4
qRT-PCR Analysis of Gene Expression in Yersinia
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qRT-PCR was performed using the SensiFastNoRox Kit (Bioline) with 25 ng/μl of the RNA samples according to the manufacturer's instructions. qRT-PCR was performed in a Rotor-Gene Q lightcycler (Qiagen). Primers used for analyzing relative gene expression purchased from Metabion and are listed in S2 Table . The gene sopB was used for normalization. Data analysis was performed with the Rotor-Gene Q Series Software. Relative gene expression was calculated as described earlier [66 (link)]. Primer efficiencies were determined experimentally using serial dilutions of genomic Y. pseudotuberculosis YPIII DNA. Primer efficiencies are: csrB: 2; csrC: 2; pnp: 2; rne: 2; sopB: 2.
5
Quantitative Real-Time PCR Analysis of Gene Expression
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Total RNA was isolated from logarithmically grown cultures using RNeasy mini kit (Qiagen) as described previously [115 (link)]. Reverse transcription and quantitative real-time PCRs (qRT-PCR) were performed using the SensiFast SYBR No-ROX One Step kit (Bioline) in a Rotor-Gene Q lightcycler (Qiagen). Relative changes in mRNA levels were analysed according to Livak and Schmittgen [116 (link)] and normalized against the transcription levels of multiple reference genes according to the method described by Vandesompele et al. [117 (link)]. The reference genes gyrB and gmk were used as previously described [52 (link)].
6
RNA Extraction and RT-qPCR Protocol
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RNA was isolated using the RNA-Bee (Tel-Test, Inc.) reagent. Contaminating DNA was removed by incubation with amplification grade DNase I (Invitrogen) and cDNA was prepared from random-primed RNA using Superscript III (Invitrogen) as per the manufacturer's instructions. Real-time quantitative PCR (qPCR) of cDNA or genomic DNA samples was performed using SensiMix SYBR Kit (Bioline) on a Rotor-Gene Q light cycler (QIAGEN). For quantitation, standard curves were created using dilutions of genomic BCBL1 DNA over a range of at least 10000×. The sequences of all primer pairs used in this study are given in Table S1 .
7
Quantifying Bacterial Gene Expression
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Strains were grown under agitating growth conditions in LB medium and total RNA isolation was performed using the RNeasy Mini kit (Qiagen). For removal of genomic DNA, RNA was treated with DNase using the TURBO DNA-free kit (Ambion). Reverse transcription and quantitative real-time PCRs (qRT-PCR) were performed using the SensiFast SYBR No-ROX One Step kit (Bioline) in a Rotor-Gene Q Lightcycler (Qiagen). Relative changes in mRNA levels were analyzed according to Pfaffl64 (link) and normalized against the transcription levels of the reference genes gyrB, gmk and rpoD28 (link).
8
RNA Extraction and Real-Time RT-PCR Assay
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For the extraction of RNA, cells were incubated with RNAlater (Ambion) for 1 min, washed with water and lysed in QIAzol (Qiagen). RNA was extracted according to manufacturer's instructions. 1 μg of total RNA was used to synthesize cDNA with Superscript II Reverse Transcriptase (Invitrogen LT) and oligo(dT) primers (Invitrogen LT) according to the recommended protocol.
Semi-quantitative RT-PCR was performed with GoTaqTM DNA polymerase (Promega) according to the instructions of the producer. For quantitative real-time RT-PCR a Rotor-Gene®Q LightCycler (Qiagen) detecting Rotor Gene SYBR Green (Qiagen) was used. All values were normalized to β-actin mRNA as internal standard. Oligonucleotide primers and annealing temperatures used for gene amplification are listed in Supplementary TableS2 .
Semi-quantitative RT-PCR was performed with GoTaqTM DNA polymerase (Promega) according to the instructions of the producer. For quantitative real-time RT-PCR a Rotor-Gene®Q LightCycler (Qiagen) detecting Rotor Gene SYBR Green (Qiagen) was used. All values were normalized to β-actin mRNA as internal standard. Oligonucleotide primers and annealing temperatures used for gene amplification are listed in Supplementary Table
9
Quantitative Gene Expression Analysis
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Total RNA was extracted from cells, cDNA synthesized and realtime reverse transcription polymerase chain reaction performed as described 5, (link)23 (link) using SYBR Green and a Rotor-GeneQ LightCycler (Qiagen). Primers for gene amplification are listed in Online Table III.
10
Kidney tissue gene expression analysis
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Total RNA was extracted from mouse kidney tissue using the Nucleospin RNA Extraction kit, according to the manufacturer's instructions (Macherey-Nagel). One microgram of RNA was then retro-transcribed using the qScript cDNA Supermix (Quanta Biosciences). Quantitative real-time PCR were performed with the 2X Perfecta SYBR Green Mix (Quanta Biosciences), using the Rotor-Gene Q Lightcycler (Qiagen). For each primer pair used, PCR efficiency was determined and integrated to calculate relative mRNA quantity against a standard curve. We evaluated six housekeeping genes (β-actin, hypoxanthine phosphoribosyl-transferase 1 (HPRT), Ribosomal Protein L5 (RLP5), non-POU-domain-containing, octamer binding protein (Nono), HydroxyMethylBilane Synthase (HMBS) and vascular endothelial growth factor A (VEGF-A) to determine the reference gene most suitable to normalize the Cp value of IL-33 and MCP-1 genes with the same set of kidney samples from euglycemic and hyperglycemic WT mice. Results were then normalized against VEGF-A as a loading control (identified as the most stably expressed gene according to the averaged expression stability values). See Supplemental Table for primer sequences and accession numbers.
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