After serological tests were completed, 260 seronegative cattle were selected for the detection of PI animals. Ear notch biopsies were collected and frozen at − 80 °C until used. About 1 cm3 of tissue was then fragmented in 1 mL of sterile PBS and centrifuged at 13.000×g for 5 min. RNA was extracted from the supernatant using a RNeasy Mini Kit (QIAGEN®, Germany) and stored at − 80 °C until used. Two steps RT-PCR was performed for the detection of the virus, in which the retrotranscription step was carried out with random primers, (Invitrogen® USA), MLV-RT (Invitrogen®, USA), dNTPs (Invitrogen®, USA) and RNA out (Invitrogen®, USA). After this, it was used the cDNA for the PCR using primers previously described [35 (link)] directed to the 5’UTR of the virus with an expected product of 288 bp with a PCR master mix (Roche ®). The sensitivity of the PCR was tested by cloning the PCR product on a pELMO vector (See section below) [36 ]. The results were observed on 1.5% agarose gels prepared in 1× TAE (Biorad®, USA) and dyed with 1× HydraGreen fluorescent inter-calating dye (ACTGene, USA). DNA extracted from a blood sample of an infected animal was used as positive control for BVDV and RNase- and DNase-free water as negative amplification control.
Rnaout
Manufactured by Thermo Fisher Scientific
Sourced in Argentina
RNAout is a reagent designed for the isolation and purification of RNA from a variety of biological samples. It utilizes a guanidinium thiocyanate-phenol-chloroform extraction method to effectively separate RNA from other cellular components.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Pcr master mix, by Roche (1 mentions)
Mlv rt, by Thermo Fisher Scientific (1 mentions)
Dntps, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Random primer, by Thermo Fisher Scientific (1 mentions)
Deoxyribonucleotide triphosphate, by Thermo Fisher Scientific (1 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Spectrostar nano, by BMG Labtech (1 mentions)
Random primer, by Promega (1 mentions)
Moloney murine leukemia virus reverse transcriptase, by Promega (1 mentions)
M mlv rt, by Thermo Fisher Scientific (1 mentions)
4 protocols using rnaout
1
BVDV Persistently Infected Cattle Detection
2
RNA Isolation and cDNA Synthesis
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To avoid putative genomic DNA contamination, RNA samples were treated with DNAse (Invitrogen) according to the manufacturer's instructions and assessed for quantity with SPECTROstar Nano (BMG Labtech), aliquoted and stored at -80 °C. First-strand cDNA was synthesized using a master mix (Moloney Murine Leukemia Virus (MMLV) buffer, dithiothreitol, RNAout, MMLV reverse transcriptase, deoxyribonucleotide triphosphate and random primers (Invitrogen)). The reverse transcription conditions were as described previously (Rodríguez et al., 2013 (link)(Rodríguez et al., , 2011;; (link)Marelli et al., 2014) (link). Briefly, they consisted of 10 min of annealing at 25 °C, 50 min of cDNA synthesis at 37 °C and 15 min of inactivation at 70 °C.
3
Total RNA Extraction and cDNA Synthesis
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Total RNA was individually extracted using TRIZOL reagent (Life Technologies, Grand Island, NY, USA) according to the manufacturer's instructions. The concentration and purity of total RNA was determined by measuring the optical density at 260 and 280 nm. All samples were precipitated with ethanol, and then dissolved in distilled water, and their quality was verified by gel electrophoresis. Equal quantities (1 g) of total RNA were reverse-transcribed into cDNA with Moloney Murine Leukemia Virus reverse transcriptase (300 units; Promega, Madison, WI, USA) using 200 pmol of random primers (Promega). Twenty units of ribonuclease inhibitor (RNAout; Invitrogen Argentina, Buenos Aires, Argentina) and 100 nmol of a deoxynucleotide triphosphate (dNTP) mixture were added to each reaction tube at a final volume of 30 l of 1x reverse transcriptase buffer. Reverse transcription was performed at 37 • C for 90 min and at 42 • C for 15 min. Reactions were stopped by heating at 80 • C for 5 min and cooling on ice.
4
Uterine Horn RNA Extraction and cDNA Synthesis
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Individual uterine horn samples from each experimental group were homogenized in TRIzol reagent (Invitrogen) and total RNA was extracted according to the manufacturer's instructions. The concentration and purity of total RNA was determined by measuring the optical density at 260 and 280 nm. All samples were precipitated with ethanol, dissolved in distilled water, and then stored at -80°C until needed. Equal quantities (1 μg) of total RNA were reverse-transcribed into cDNA with Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT; 300 units; Promega) using 200 pmol of random primers (Biodynamics, Buenos Aires, Argentina). Twenty units of ribonuclease inhibitor (RNAout, Invitrogen Argentina) and 100 nmol of a deoxynucleotide triphosphate mixture were added to each reaction tube at a final volume of 30 μL of 1× MMLV-RT buffer. Reverse transcription was performed at 37°C for 90 min and at 42°C for 15 min. Reactions were stopped by heating at 80°C for 5 min and cooling on ice. Each reverse-transcribed product was diluted with RNase-free water to a final volume of 60 µL.
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