Kappa sybr fast qpcr kit

Origene’s qStar mRNA detection system (Origene, Rockville, MD) was used in the quantification of AXL mRNA in cord blood in NEST subjects. qPCR primers for the major AXL transcript (#HK228780) and its corresponding copy number standard (#HK201002) were designed by qStar. All measurements of expression were conducted in duplicate in cord blood samples from 235 participants in the NEST cohort. AXL mRNA was isolated from stored PAXgene tubes of cord blood using the PAXgene blood miRNA isolation kit (Qiagen, Valencia, CA). First strand cDNA conversion of mRNA was performed using Origene’s cDNA synthesis kit (#NP100042). qPCR reactions were run with Kappa Sybr Fast qPCR kit (# KK4604; KapaBiosystems, Boston, MA) in the ABI 7900HT thermocycler (Thermofisher). Ten percent repeats were included to evaluate reproducibility.

Total RNA that was extracted for microarray from the clinical subjects was also used for RT-PCR validation. RT-PCR was performed with Agilent Brilliant III Ultra-Fast RT-PCR reagent (cat#600884, Agilent Technologies, Santa Clara, CA, USA), using Agilent AriaMX real-time PCR instruments. The relative mRNA expression levels were quantified using the C(t) method. For in vitro assays, total RNA was isolated from cells using the Trizol reagent (Invitrogen, Waltham, MA, USA) according to the manufacturer’s protocol. A total of 1 µg of RNA was reverse transcribed using the Bio-Rad iScript cDNA synthesis kit (cat#1708890, Bio-Rad, Hercules, CA, USA), and quantitative real-time PCR was performed using the Kappa Sybr Fast qPCR kit (cat#KK4601, Kapa Biosystems Pty (Ltd.), Cape Town, South Africa) using the Bio-Rad CFX96 system. Relative mRNA expression levels were quantified using the C(t) method. The results were normalized to housekeeping human β-actin. The details of the primers used are described in Table S3.

Following assessment of the quality of total RNA using Agilent 2100 bioanalyzer and RNA Nano Chip kit (Agilent Technologies, CA), rRNA was removed from 2.5 µg of RNA with Ribo-Zero rRNA removal kit for Gram-negative bacteria (Epicentre Biotechnologies, WI). To generate directional signal in RNA seq data, libraries were constructed from first strand cDNA using ScriptSeq v2 RNA-Seq library preparation kit (Epicentre Biotechnologies, WI). Briefly, 50 ng of rRNA-depleted RNA was fragmented and reverse transcribed using random primers containing a 5′ tagging sequence, followed by 3′end tagging with a terminal-tagging oligo to yield di-tagged, single-stranded cDNA. Following purification by a magnetic-bead based approach, the di-tagged cDNA was amplified by limit-cycle PCR using primer pairs that anneal to tagging sequences and add adaptor sequences required for sequencing cluster generation. Amplified RNA-seq libraries were purified using AMPure XP System (Beckman Coulter). Quality of libraries were determined via Agilent 2100 Bioanalyzer using DNA High Sensitivity Chip kit, and quantified using Kappa SYBRFast qPCR kit (KAPA Biosystems, Inc, MA). 50 bp sequence reads were generated using the Illumina HiSeq 2000 platform.

Following DNase treatment and cleanup of RNA from isolated cells using Zymo RNA cleanup and concentrator kit (Zymo research, CA) quality assessment was performed on Agilent 2100 bioanalyzer with RNA Pico chip kit (Agilent Technologies, CA). RNA integrity score ≥6 was used for cDNA library preparation from each sample. 10 ng of total RNA was used to generate RNA-Seq libraries using SMARTer stranded total RNA-Seq kit-Pico input mammalian kit (Clontech Laboratories Inc., CA). Briefly, total RNA was converted to cDNA using SMART^® (Switching Mechanism at 5′ end of RNA template) and locked nucleic acid (LNA) technology included as part of the template-switching oligo (TSO). Ribosomal cDNA (originating from rRNA) was then cleaved by ZapR in the presence of mammalian-specific R-probes. The library fragments originating from non-rRNA molecules were enriched via PCR amplification. Quality of the libraries were assessed via Agilent 2100 Bioanalyzer using DNA High Sensitivity Chip kit, and quantified using Kappa SYBR^®Fast qPCR kit (KAPA Biosystems, MA). 151 bp sequence paired end reads were generated using the Illumina HiSeq 4000 platform (Illumina, CA).