A total of 23 mice were used in replicate experiments. The 1064 and 1270 nm CW laser were irradiated at 5 and 2 W/cm2, respectively, at four spots on the back of the dehaired mice, without overlap. Six hours after irradiation, 5 mm diameter skin including the irradiated area was stripped and soaked in RNAlater Stabilization Solution (Thermo Fisher Scientific, MA, USA). The collected skin was shredded into strips of tiny pieces using a pair of scissors, and homogenized in TRIzol Reagent (Thermo Fisher Scientific, MA, USA), using a Bullet Blender (NEXT ADVANCE, NY, USA). RNA extraction and its reverse transcription to cDNA were performed as described in our previous report (15 (link)). Primers used were Ccl2, Ccl20, Nfkb1, and Nfkb2 (RT2 qPCR Primer Assays; QIAGEN, Venlo, Netherlands) with guaranteed amplification efficiency, and real-time RT-PCR was performed using a LightCycler 480 System (Roche, Basel, Switzerland). The relative expression levels of each chemokine were compared using the delta-delta Ct method with Actb (RT2 qPCR Primer Assays; QIAGEN, Venlo, Netherlands) as the reference gene (18 (link)). We had earlier confirmed the stable expression of Actb with or without laser irradiation.
Rt2 qpcr primer assay
Manufactured by Qiagen
Sourced in Germany, United States, Netherlands, Spain
RT2 qPCR Primer Assays are pre-designed primer sets for quantitative real-time PCR (qPCR) experiments. They are designed to amplify target genes of interest with high specificity and efficiency.
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Lab products found in correlation
Rneasy mini kit, by Qiagen (28 mentions)
Rt2 first strand kit, by Qiagen (26 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (7 mentions)
Rt2 sybr green rox qpcr mastermix, by Qiagen (7 mentions)
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Rt2 sybr green mastermix, by Qiagen (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rt2 sybr green qpcr mastermix, by Qiagen (4 mentions)
Itaq universal sybr green supermix, by Bio-Rad (4 mentions)
Rneasy plus mini kit, by Qiagen (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Rneasy kit, by Qiagen (4 mentions)
Icycler thermal cycler, by Bio-Rad (3 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (3 mentions)
Quantstudio 12k flex real time pcr system, by Thermo Fisher Scientific (3 mentions)
Quantstudio 3, by Thermo Fisher Scientific (3 mentions)
Rt2 first strand synthesis kit, by Qiagen (3 mentions)
Rneasy micro kit, by Qiagen (3 mentions)
High capacity cdna archive kit, by Thermo Fisher Scientific (3 mentions)
110 protocols using rt2 qpcr primer assay
1
Laser-Induced Skin Transcriptome Changes
2
Quantification of PAINT Expression in PCa
3
Molecular Analysis of Spinal Cord Tissues
4
RNA Expression Analysis of Neuroinflammation
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For molecular analyses, animals were killed on day 21 after surgery. Total RNA from the lumbar spinal cord was extracted using the GeneAll Hybrid-R kit (GeneAll Biotecnology, Seoul, Korea). Next, cDNA was synthesized from 1 µg RNA using a RT 2 first strand cDNA Synthesis Kit (Qiagen, Valencia, CA). Real-time polymerase chain reactions (PCRs) were conducted using RT 2 qPCR Primer Assays and RT 2 SYBR Green ROX mastermixes (Qiagen). The RT 2 qPCR Primer Assays for mouse included toll-like receptor-4 (TLR4), interleukin (IL)-1β, nacht domain-, leucine-rich repeat-, and pyrin domain-containing protein 3 (NALP3), and β-actin. The PCR component mix for each reaction was a 25 μL final volume of 12.5 μL RT 2 SYBR green mastermix (Qiagen), 1 μL of diluted template, 1 μL RT 2 qPCR Primer Assay (Qiagen), and 10.5 μL of RNase-free water. Real-time PCR amplification was performed on an ABI 7900HT sequencing detection system in triplicate. To validate the primer sets used, we performed dissociation curves to document single-product formation, and agarose gel analysis was conducted to confirm the size. The data from real-time PCR experiments were analyzed by the comparative cycle threshold (CT) method.
5
Quantitative Real-Time PCR Reaction
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The quantitative real-time PCR reaction mix for one PCR tube was composed referring to the RT2 qPCR Primer Assays protocol (SABiosciences) as follows: 12.5 μL RT2 SYBR Green qPCR Master Mix, 8.5 μL ddH2O, 3 μL Template cDNA (27 ng) and 1 μL 10 μmol/L PCR primer pair stock.
As a negative control DNA was replaced by H2O. The primer pairs SYBR® Green Human RELA and SYBR® Green Human HPRT 1 (RT2 qPCR Primer Assay, SABiosciences) were used for targeting the gene of interest (GOI) RelA and the housekeeping gene (HKG) HPRT (Table 6 ).
As a negative control DNA was replaced by H2O. The primer pairs SYBR® Green Human RELA and SYBR® Green Human HPRT 1 (RT2 qPCR Primer Assay, SABiosciences) were used for targeting the gene of interest (GOI) RelA and the housekeeping gene (HKG) HPRT (
6
Quantifying mRNA Expression in Human and Mouse Kidneys
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Total RNA was isolated from HK-2 cells, using Direct-zol™ RNA MiniPrep supplied by Zymo Research (Irvine, CA), or mouse kidneys, using NucleoSpin RNA/Protein (Macherey-Nagel, Duren, Germany) followed by RNeasy® Mini Kit (QIAGEN, Germantown, MD). The OD ratio (optical density at 260 nm/280 nm) of RNA was always higher than 1.9. Reverse transcription was performed according to the manufacturer on 1.0 μg total RNA using iScript™ cDNA Synthesis Kit (Bio-Rad). RT2 qPCR Primer Assay [human (HK-2 cells) and mouse (kidneys) primers from QIAGEN] were used to quantify the mRNA expression of heme oxygenase 1 (HO-1) and heat shock protein 70 (Hsp70). Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (human and mouse kidney, respectively, RT2 qPCR Primer Assay from QIAGEN). Data are presented as columns, displaying mean ± SEM, for in vitro data and box plots, displaying medians and 25th and 75th percentiles, for in vivo data. The fold change values were calculated by normalizing against control samples from untreated cells or animals (controls). Expression was analyzed using iTaq™ Universal SYBR® Green Supermix (Bio-Rad). Amplification was performed as described by the manufacturer (Bio-Rad) for 40 cycles in an iCycler Thermal Cycler (Bio-Rad) and data analyzed using iCycler iQ Optical System Software (Bio-Rad).
7
Gene Expression Analysis Protocols
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Gene expression analysis was performed using SYBR green based assays (Applied Biosystems, USA) for HLA-G, interferon-γ (IFN-γ), markers of proliferation and differentiation- ki-67, keratin 18 and cyclin D1, immune checkpoint molecules- interleukin 10 (IL-10), tumor growth factor-β (TGF-β), programmed cell death protein 1 (PD1), suppressor of cytokine signaling 1 and 3 (SOCS1 and SOCS3) on StepOne Plus Real-Time PCR System (Applied Biosystems, United States). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the endogenous control for normalization of expression levels. Histopathologically confirmed adjacent normal tissues were used as the calibrators for mRNA quantification by comparative CT method. Primers for IL-10 and TGF-β were designed using Primer Express software while for other genes RT2 qPCR Primer Assays (Qiagen, Hilden, Germany) were used as shown in the Supplementary Table 1 . Real-time PCR was carried out in 10 μL of reaction volume consisting of 1X SYBR green PCR master mix, 0.4 μM of each primer, 200 ng of cDNA and water to adjust the volume. The relative expression of the target genes was determined using the formula 2−ΔΔct method.
8
Gene Expression Analysis Using RT-qPCR
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cDNA was generated from samples as previously described.6 (link) Expression analysis was performed using SYBR Green chemistry on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). In addition to the regulatory factors, expressions were also checked for IL-10, TNF-α and IL-1β. Primers for some of the genes were designed using Primer Express software (Applied Biosystems) (Table 2 ) while for the other genes commercially available RT2 qPCR Primer Assays (Qiagen, Hilder, Germany) were used. IDT Oligoanalyzer tool (Integrated DNA Technologies, Coralville, IA, USA) was used to screen designed primers for secondary structure formation. Real-time PCR was carried out in 10 μl reaction volume consisting of 1 × SYBR Green PCR master mix, 0.4 μm of each primer, 100 ng cDNA and water to adjust the volume using standard conditions. Gene expression analysis using TaqMan-based assay (Applied Biosystems) was performed only for CTLA4 gene. Expression levels were normalised using GAPDH as the endogenous control while controls were used as the calibrator. The relative fold changes were calculated based on the 2−ΔΔCT method. Melt curve analysis was performed to confirm the presence of specific amplification products.
9
Kinetic Analysis of IL-8 Expression
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Cells were stimulated with 20 µM FnIII-1c for 2 h. 10 µg/ml actinomycin D (Enzo Life Sciences) was added to stop further transcription in the presence or absence of 20 µM MK-2 Inhibitor III. Total RNA was extracted with RNeasy extraction kit (Qiagen) at 0, 30 and 60 min after treatment with actinomycin D. The integrity and purity of the RNA was assessed by denaturing agarose gel electrophoresis (clean and sharp 28S, 18S and 5S bands), as well as spectrophotometry via Nanodrop. 1.5 µg of RNA from each sample was reverse-transcribed using the RT2 First Strand Kit (Qiagen) according to the manufacturer's instructions. IL-8 and β-actin RT2 qPCR Primer Assays (Qiagen) were utilized for cDNA amplification. Quantitative PCR was performed using RT2 SYBR Green Mastermix (Qiagen) in a MyiQ Cycler System (Bio-Rad Laboratories). The 2∧−ΔΔCt method was utilized to measure the relative expression levels of IL-8 and β-actin. The results take into account the values for six separate experiments.
10
Quantification of mRNA and miRNA Expression
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Total RNA was isolated from primary T cells or Jurkat cells using the RNeasy Mini Kit (Qiagen). 100 ng-200 ng reverse transcription reactions for cDNA synthesis were carried out using the miScript RT Kit (Qiagen) according to the protocol for subsequent quantification of miRNAs and mRNAs.
Real-time PCR (RT-PCR) analysis of gene (mRNA) and miRNA expression was carried out using Quantitect SYBR Green Primer Assays (Qiagen, for mRNAs) and miScript SYBR Green Primer Assays (Qiagen, for miRNAs) and the Bio- Rad CFX96 real-time cycler. For some gene expression analysis, we used the RT2 qPCR Primer Assays (Qiagen). βeta-2 microglobulin (β2M) was used as an internal control for gene expression. The small nuclear RNA, U6, was used as an internal control for miRNA expression. For extravesicular miRNA expression, C. elegans miR-39 which detects the Spike-In Control added to samples during EV RNA isolation was used for normalization of RT-PCR results for miRNA expression. For data analysis, we used the ΔΔCT method of relative quantification.
Real-time PCR (RT-PCR) analysis of gene (mRNA) and miRNA expression was carried out using Quantitect SYBR Green Primer Assays (Qiagen, for mRNAs) and miScript SYBR Green Primer Assays (Qiagen, for miRNAs) and the Bio- Rad CFX96 real-time cycler. For some gene expression analysis, we used the RT2 qPCR Primer Assays (Qiagen). βeta-2 microglobulin (β2M) was used as an internal control for gene expression. The small nuclear RNA, U6, was used as an internal control for miRNA expression. For extravesicular miRNA expression, C. elegans miR-39 which detects the Spike-In Control added to samples during EV RNA isolation was used for normalization of RT-PCR results for miRNA expression. For data analysis, we used the ΔΔCT method of relative quantification.
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