Kapa sybr fast one step qrt pcr kit

A real-time qRT-PCR was performed on RNA samples using gene-specific oligonucleotide forward (F) and reverse (R) 5′–3′ primers (P21F: CAACAGCATGCCTAGCAGAA and P21R: TCCATGCTTACAACCACCAA, P30F: ATTTCCGCATTTGACTACGC and P30R: GGCCAATTTCTTGGATCTGA, P33F: GTAACATTGCCTGGCCTCAT and P33R: TTCCAAGAGCTCGAACAGGT, P37F: TGCCGGTCAATTTCCATTAT and P37R: CATGTTTGCCTCGGAAGAAT, P14F: GCGAATCACGGAGTGGTACT and P14R: CCATGGAAACCCAATTTTTG), the Kapa SYBR Fast One-Step qRT-PCR Kit (Kapa Biosystems, Wilmington, MA, USA), and the Rotor-Gene Real-Time PCR Detection System (Qiagen Inc. Valencia, CA, USA). A dissociation curve was run at the end of the reaction to ensure that only one amplicon was formed and that the amplicons denatured consistently at the same temperature range for every sample. The mRNA levels were normalized against elongation factor 1 α (EF1α) as described previously [29 (link)] using the genNorm method (ddCT method as implemented by Bio-Rad iQ5 Standard Edition, Version 2.0) [30 (link)]. Normalized Ct values were compared between unfed and fed lice by Student’s t-test with unequal variance (p = 0.05; n = 3 biological replicates).

Total RNA was obtained from WT and HGPS cells using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s instructions. The RNA yield and purity were assessed in a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Inc.). The qRT-PCR assays were carried out on the Step One Plus Real Time PCR System (Applied Biosystems, Foster, CA, MA), using the KAPA SYBR Fast One Step qRT-PCR kit (Kapa Biosystems; Wilmington, MA, USA) and a standard protocol for PCR cycling. The PGC-1α expression levels were quantified using the comparative 2^ΔΔct method and β-actin as an endogenous control. Primer sequences were as follows: PGC-1α, forward 5′-ATCCTCTTCAAGATCCTGCT-3′, reverse 5′-GACTCTCGCTTCTCATACTCTC-3′; actin; forward 5′-CAGACAGCGAAAGGATGAG-3′, reverse 5′-CAGACAGCGAAAGGATGAG-3′.

RNA from artificial skin was isolated using TRIzol Reagent (Invitrogen, USA), according to the manufacturer’s instructions. Complementary DNA synthesis and amplification were performed using the CFX96 Touch Real-time PCR Detection system thermocycler and KAPA SYBR FAST One-Step qRT-PCR Kit (KAPABIOSYSTEMS, USA), according to the manufacturers’ protocols. The primer pairs used are presented in Table 3. The level of mRNA was normalised to the levels of β-actin or TGase-1 mRNA. Subsequently, the relative mRNA expression levels were calculated using the 2^−ΔΔCt method.Table 3

Primers used for qRT-PCR analysis.

Gene	Forward	Reverse
FLG	5′-AAGGTTCACATTTATTGCCAAA-3′	5′-GGATTTGCCGAAATTCCTTT-3′
β-Actin	5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′	5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′
Caspase-14	5′-AAATGAGCAATCCGCGGTCTTTGG-3′	5′-CCGTGGAATAAACGTGCAAGGCAT-3′
Involucrin	5′-CTCCACCAAAGCCTCTGC-3′	5′-CTGCTTAAGCTGCT GCTC-3′
TGase-1	5′-TGAATAGTGACAAGGTGTACTGGCA-3′	5′-GTGGCCTGAGACATTGAGCAGCAT-3′
psoriasin-hS100A7	5′-AGACGTGATGACAAGATTGAC-3′	5′-TGTCCTTTTTCTCAAAGACGTC-3′
Koebnerisin 15S-hS100A15S	5′-CAAGTTCCTTCTGCTCCATCTTAG-3′	5′-AGCCTTCAGGAAATAAAGACAATC-3′
Koebnerisin 15L-hS100A15L	5′-ACGTCACTCCTGTCTCTCTTTGCT-3′	5′-TGATGAATCAACCCATTTCCTGGG-3′

RNA was extracted from three, independent, 10-µm section from paraffin-embedded tissues of each patient using RNeasy FFPE Kit (QIAGEN, Milan, Italy), following the manufacturer’s protocol. Real-time PCR was performed using the KAPA SYBR FAST One-Step qRT-PCR Kit (KAPA-Biosystems, Boston, Massachusetts, USA), following the manufacturer’s protocol using the CFX96™ Real-Time PCR Detection System (Bio-Rad). Each analysis was performed in duplicate and the expression level of FMR1 mRNA was normalized by ACTB mRNA.

qRT-PCR was carried out in a qTOWER Thermal Cycler (Analytik Jena, Germany) using the KAPA SYBR FAST One-Step qRT-PCR Kit and 50 ng of RNA per reaction (5 μl). The ratios of transcript abundance were calculated as the 2^–ΔCT mean average of 3 replicates, where CT indicates the level of gene expression in the specified strain relative to the expression in the control strain. The uniformly expressed gene SMc01852 [86 (link)] was used to normalize the gene expression data.

RNA from the immunoprecipitated pellets or from total cell lysates were isolated using the RNeasy kit (Qiagen, Grand Island, NY, United States). The KAPA SYBR^® FAST One-Step qRT-PCR Kit (KAPA Biosystems, Wilmington, MA, United States) was used using 1 μg of RNA as input. Primers used for amplification were:
ICAM1, VCAM1, and IL6 expression levels were calculated by the 2-ΔΔCt method. GAPDH was used as an endogenous control for data normalization. The data was also re-analyzed with 18s rRNA and did not show any difference as when GAPDH was used (data not shown). Relative enrichment of lncRNAs in RIP analysis was performed based on normalization to enrichment with just IgG.

Total RNA was isolated from monolayers using a Nucleospin RNA II kit (MACHEREY-NAGEL EURL, Hoerdt, France) according to the manufacturer’s instructions. 100 ng of total RNA extracted from healthy or psoriatic keratinocytes (until passage 1) were subjected to the first strand cDNA synthesis and QPCR analysis using the KAPA SYBR FAST One-Step qRT-PCR KIT (Kapa Biosystems, Wilmington, MA, USA). The primers (Eurofins Genomics AT GmbH) used for the detection of gene expression are listed below. Reactions were performed using a Corbett 6000 real-time PCR cycler Rotorgene. The presence of a single dissociation peak was verified by melt curve analysis. Relative quantification was determined using the comparative (Ct) method [59 (link)] with normalization to the housekeeping gene GAPDH.
Syndecan-1 FP: CTTCACACTCCCCACACAGA, RP: GTATTCTCCCCCGAGGTTTC, Syndecan-4 FP: TGAGGATGTGTCCAACAAGG, RP: AGGAAGACGGCAAAGAGGAT K6: FP: GTGAGGAGTGCAGGCTGAAT, RP: CATAGCCACTGGAGACGGTG, K14: FP: TCATCCAGAGATGTGACCTCC, RP: GCCTCAGTTCTTGGTGCGAA, K10: FP: TGATGTGAATGTGGAAATGAATGC, RP: GTAGTCAGTTCCTTGCTCTTTTCA, GAPDH: FP: TGCACCACCAACTGCTTAGC, and RP: GGCATGGACTGTGGTCATGAG.