Rnaseout recombinant

After hypotonic red cell lysis from at least 20 mL peripheral blood or bone marrow total RNA was extracted using a commercially available kit (innuPREP RNA Mini Kit, Analytik Jena AG, Jena, Germany) according to the manufacturer’s instructions. cDNA was synthesized using oligo(dt)-primer, dNTP mix (Fermentas), random hexamer primers (Thermo Fisher Scientific), reaction buffer (5 × First Strand Buffer, Invitrogen), DTT (Invitrogen), RNaseOut Recombinant (40 U mL^-1 Ribonuclease Inhibitor) and M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase (Invitrogen) as described elsewhere.³⁸ (link)

Endoscopic tumor and normal adjacent rectal biopsies were collected from each patient before pCRT, according to a standard protocol approved by the local ethics committee. Briefly, each patient signed an informed consent for the use of these samples for research purposes. All biopsies underwent standardized histopathological examination based on hematoxylin-eosin staining of 5 μm frozen sections. Tumor specimens with≥ 70% malignant cells were considered for the experiment. Total RNA extraction, from at least 2 micro-biopsies, was performed using TRIZOL® Reagent (Invitrogen, Carlsbad, CA) following standard procedures from each endoscopic biopsy by sections of 20 μm thick. Total RNA was preserved in a final volume of 20 μl of DEPC water at -80°C with 1μl RNase Inhibitor (RNaseOUT Recombinant, 40 U/μl, Invitrogen). RNA quantity was measured on an ND-1000 spectrophotometer (NanoDrop Technologies) and quality was assessed by capillary electrophoresis with Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). Samples with RIN> 6.5 (RNA 6000 Series Nano Chips) and samples enriched for small nucleic acid fragments with a percentage <35% (Agilent Small RNA Kit) were selected for the microarray analysis and qRT-PCR.

After the transfection, total RNA was extracted using the RNeasy mini kit from Qiagen, and the amount of RNA was assessed by NanoDrop (Thermo Scientific, USA). The cDNA from each sample was then synthesized using 1 μg of RNA added to previously mixed 1 μL of Oligo (dT) 12–18 bp primers (Invitrogen) and 1 μL of dNTP Mix (Invitrogen), incubated for 5 min at 65°C and reaction stopped by incubation on ice for 1 min. After that the mixture was added to a premix that was made of 4 μL of 5× first single strand buffer, 1 μL of RNaseOut Recombinant, 1 μL of 0.1 M DTT, and 1 μL of Superscript III RT (all from Invitrogen). The generated cDNA was diluted and kept at −20°C.