Zymo direct zol rna miniprep kit

Total RNA was extracted from isolated EVs using miRNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions, with minor modifications. Total RNA from placental tissue was isolated using Zymo Direct-zol RNA miniprep kit (#R2050; Zymo research, Irvine, CA, USA) following manufacturer’s instructions. Briefly, the snap frozen and subsequently powdered tissue was incubated in TRIzol (#15596026; ThermoFisher Scientific, Waltham, MA, USA) for 5 min. The mixture was homogenized with a hand-held homogenizer for 1 min on ice and centrifuged at high speed for 2 min at 4°C. Collected supernatant was directly applied to Zymo spin column and centrifuged at high speed to bind RNA to silica membranes. On-column digestion of genomic DNA was performed using DNaseI (supplied with the kit) followed by washing with ethanol containing wash buffer. RNA was eluted with 10mM Tris and the concentration was measured using Qubit3 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA). The Agilent Bioanalyzer or Tape Station (Agilent, Santa Clara, CA, USA) was used to determine RNA integrity numbers (RIN) prior to library preparation.

Total RNA was purified using a Zymo Direct-zol RNA MiniPrep kit (Zymo Research). The quality and quantity of RNA samples was measured using an Agilent RNA 6000 Nano kit (Agilent) on a 2100 Bioanalyzer (Agilent) and a Nanodrop 2000 spectrophotomoter (Thermo Scientific). Libraries for small RNA sequencing were prepared (normal colon mucosa, NM, n=2; LG-IEN, n=5; HG-IEN, n=5) using 1 μg total RNA with TruSeq Small RNA Library Prep Kit (Illumina) according to the manufacturer's instructions. Twelve samples were pooled and after validation (Agilent 2100 Bioanalyzer) run on MiSeq system (Illumina) using MiSeq Reagent Kits v2(Illumina) at 1×50 bp read configuration to generate FASTQ files.

Total RNA was extracted with TRI Reagent (Zymo Research, R2050-1-200). RNA was then isolated using the Zymo Direct-zol RNA Miniprep Kit (Zymo Research, catalog number R2053) and reverse-transcribed using Bio-Rad iScript Reverse Transcription Supermix (Bio-Rad, catalog number 1708841) in a Bio-Rad C1000 Touch Thermal Cycler. Quantitative mRNA expression was determined by real-time qPCR using iTaq Universal SYBR Green Supermix (Bio-Rad, catalog number 172–5121).
qPCR primer sequences used are as follows:

Pellets of sorted cells were collected for total RNA extraction using Zymo Direct-zol RNA Miniprep Kit (Zymo Research, Irvine, CA, USA, R2063) following manufacturer’s instructions. RNA quality was verified by Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). Indexed cDNA libraries were prepared using the Illumina^® Stranded mRNA Prep Ligation kit (Illumina, San Diego, CA, USA, 20040532) following manufacturer’s instructions. The cDNA libraries were then submitted to the Next Generation Sequencing Core at UT Health San Antonio for high throughput sequencing analysis using an Illumina NovaSeq 6000 System.

RNA was extracted using a Zymo Direct-Zol RNA Miniprep kit (Zymo Research, R2051). cDNA was prepared from 25 ng of RNA using oligoDT primers in a 20 µl reaction using a High Capacity cDNA Reverse Transcription Kit (ThermoFisher, 4368814). qRT-PCR reactions were performed in real time using 5 µl PowerUp SYBR Green Master Mix (ThermoFisher, A25741), 0.8 µl of 10 µM forward and reverse primers, 1.4 µl of nuclease-free water, and 2 µl of diluted cDNA. PCR was performed using a QuantStudio Real Time PCR System (Applied Biosystems) with cycling parameters: 50°C (2 min), 95°C (10 min) then 40 cycles of 95°C (15 s), and 60°C (1 min). Each reaction was performed in quadruplicate. Quantitation was relative to rpl13 and used the ∆∆C_T relative quantitation method in which fold changes are calculated as 2^−∆∆CT. The efficiency of amplification was verified to be close to 100% with a standard curve of RNA dilutions.