Rneasy minielute kit

RNA was extracted from peripheral whole blood samples using the PAXgene Blood miRNA kit (PreAnalytix GmbH, Switzerland). The extractions were performed according to the manufacturer’s protocol. The RNeasy MiniElute kit (QIAGEN, Manchester) was used to obtain the required concentration and volume for the Globin mRNA reduction procedure.
Globin mRNA was removed from the RNA samples using the Human GLOBINclear kit (Ambion Inc., Texas, USA). The purity and the concentration of the globin-cleared samples were assessed using the Nano-drop ND-1000 spectrophotometer (Willmington, USA). The samples were stored at −20°C, according to the GLOBINclear manufacturer’s protocol [68 ]. The quality of all samples was analysed with the Agilent 2100 Bioanalyzer using the Agilent RNA Nano kit (Agilent, Santa Clara, USA). Samples with a RNA integrity number (RIN) of above seven were used for whole genome microarray using the Illumina HumanHT-12 v4 BeadChip. Both techniques were performed at Cambridge Genomic Services (Cambridge, UK).

Total RNA was isolated using TRIzol^® (Invitrogen). The dried pellet was cleaned with the RNeasy MiniElute Kit (Qiagen, Hilden, Germany). RNA-concentration was measured on a NanoDrop^® Spectrophotometer (Peqlab, Erlangen, Germany). Real-time RT-PCR was conducted to quantify gene expression (primer sequence: hRARRES1; Forward: 5′-AGGTGTCACACTACTACTTGG-3′; Reverse: 5′AGCTGTTGACAGTGGTACTTC-3′). One microgram of total RNA was reverse-transcribed using the Transcriptor-cDNA-Kit (Roche, Grenzach-Wyhlen, Germany). PCRs were carried out in a Mastercycler^® ep-realplex (Eppendorf, Germany). Data were analyzed according to the comparative C_T method and normalized for Cyclophilin expression in each sample. PCR primers were ordered from Thermo Fisher, Germany (Hs00161204_m1).

ZFN RNA and TALEN RNA were synthesized with Ambion’s mMESSAGE mMACHINE T7 Ultra and T3 kits, respectively using XbaI-linearized ZFN and SacI-linearized TALEN plasmids as templates. Following poly-A-tailing of the capped transcript with Epicentre’s poly (A) polymerase tailing kit, the in vitro produced mRNA was purified with Qiagen’s RNeasy mini elute kit.

The total RNA from the ovaries was extracted by using TRIzol reagent (Invitrogen, Carlsbad, CA), and the RNA integrity number (RIN) value of all the samples was greater than 8. RNA was purified with an RNeasy MiniElute Kit according to the manufacturer’s instructions (Qiagen, Valencia, CA). A TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) was used for the library construction. The libraries were then assessed with an Agilent Technologies 2100 Bioanalyzer and an Agilent High Sensitivity DNA Kit (Agilent Technology, US). The pooled libraries were sequenced on an Illumina HiSeq2000 (Illumina, San Diego, CA). After sequencing, the indexed adapter sequences were trimmed by using CASAVA software (Illumina).

Total RNA was isolated by using TRIzol (Invitrogen). The dried pellet was cleaned with the RNeasy MiniElute Kit (Qiagen). RNA concentration was measured on a NanoDrop Spectrophotometer (Peqlab). Real-time RT-PCR was conducted to quantify gene expression. Total RNA (1 μg) was reverse transcribed by using the Transcriptor cDNA Kit (Roche). PCRs were carried out in a Mastercycler ep-realplex (Eppendorf). Data were analyzed according to the comparative CT method and normalized for cyclophilin expression in each sample.

RNA transcription profiles from experimental and control samples were created using a microarray assay. RNA samples were analyzed using a NanoDrop 2000 (Thermo Fisher, Waltham, MA) and quality was assessed with an Agilent 2100 Bioanalyzer (Agilent, Wilmington, DE). RNA integrity was expressed as RNA integrity number (RIN) values and only samples with values greater than 10 were used. The Low RNA Input Linear Amplification kit with one-color (Agilent, Wilmington, DE) was used to produce fluorescent cRNA. Labeled cRNA was then purified using the RNeasy Mini Elute kit (Qiagen, Valencia, CA). Fluorescent cRNA samples were then hybridized onto Whole Human Genome 4 × 44 K microarrays GeneChips (Affymetrix, Santa Clara, CA). Four replicates were performed with array hybridization conducted for 17 h at 65 °C. Slides were then scanned with an Agilent microarray scanner (G2565BA) using Feature Extraction software (v 9.5.1, Agilent). GeneSpring GX 10.0.2 software (Agilent Technologies) was used for statistical analysis, background correction, normalization and summary of expression measure.