Sixteen 6-month-old castrated boars from populations of Tibetan pigs (TPs, n = 8) and Yorkshire pigs (YPs, n = 8) that were raised at the experimental farm of the Tibet Agriculture and Animal Husbandry College (Linzhi, 3000 m above sea level) were slaughtered and sampled. Cardiac tissue samples were immediately frozen in liquid nitrogen and stored at -80°C until RNA extraction. Total RNA for miRNA sequencing was extracted using TRIzol reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol. Extract quality was checked using a NanoDrop™ Biophotometer 2000 (Thermo Fisher Scientific Inc., West Palm Beach, FL, USA); a 260/280 nm absorbance ratio of 1.8–2.0 indicated a pure RNA sample. Equal quantities of the RNA extracted from the cardiac tissue of four pigs in each population were pooled into one. Thus, we had two duplicate samples in Tibetan pigs, two duplicate samples in Yorkshire pigs. Specific details are provided in S1 Table . The experiments were approved by the animal welfare committee of the State Key Laboratory for Agro-Biotechnology of the China Agricultural University (Approval number XK257). Pig farming at Linzhi was permitted, and the field study did not involve endangered or protected species.
Nanodrop 2000 biophotometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The NanoDrop 2000 Biophotometer is a compact and versatile spectrophotometer designed for the quantification and analysis of nucleic acids and proteins. It utilizes a patented sample retention system that requires only 1-2 microliters of sample to perform measurements, making it well-suited for applications where sample volume is limited.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Enhanced bca protein assay kit, by Beyotime (2 mentions)
Sds lysis buffer, by Beyotime (2 mentions)
Trizol reagent, by Roche (1 mentions)
Primescript rt reagent kit with gdna eraser perfect real time, by Takara Bio (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Fastquant reverse transcriptase kit, by Tiangen Biotech (1 mentions)
Improm iitm reverse transcriptase, by Promega (1 mentions)
Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Rna 6000 nano labchip kit, by Agilent Technologies (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (1 mentions)
Hiscansq platform, by Illumina (1 mentions)
Truseq rna sample preparation kit set a, by Illumina (1 mentions)
Fastquant rt kit, by Tiangen Biotech (1 mentions)
8 protocols using nanodrop 2000 biophotometer
1
Cardiac miRNA Profiling of Tibetan and Yorkshire Pigs
2
RNA Extraction and Reverse Transcription
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Total RNA from each tissue was extracted using Trizol reagent (Tri Pure Isolation Reagent, Roche, Carlsbad, CA, USA), following the manufacturer’s instructions. The concentration and quality of RNA and DNA were examined using a Nano Drop™ Bio Photometer 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The OD 260/280 nm values were within the appropriate range of 1.8–2.1, indicating that the purity of the RNA samples was good. Additionally, the 28S and 18S bands were clear and were not degraded by 1% agarose gel electrophoresis, indicating that the RNA integrity and quality were good and that follow-up tests could be carried out. Additionally, a Prime Script RT reagent kit with gDNA Eraser (Perfect Real Time) (TaKaRa Bio Inc., Shiga, Japan) was used for cDNA synthesis. After reverse transcription, cDNA was stored at −20 °C.
3
Genomic DNA and RNA Isolation Protocol
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Genomic DNA was isolated from ear tissue using the standard phenol/chloroform extraction method, dissolved in a TE solution, and stored at −20 °C. Total RNA was extracted from tissues and cells using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and its quality and integrity were verified using a NanoDrop 2000 Biophotometer (Thermo Fisher Scientific Inc, West Palm Beach, FL,USA) and via electrophoresis. RNA samples (2 μg) in a 20 μL reaction volume were reverse transcribed to cDNA using the FastQuant Reverse Transcriptase Kit (TIANGEN, Beijing, China).
4
Genomic DNA and RNA Extraction from Mammalian Tissues
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Genomic DNA was isolated from the ear tissues using the method described by Sambrook et al. [10 ], dissolved in Tris-EDTA (TE) buffer, and stored at −20 °C.
Total RNA was extracted from tissues with TRIZOL® Reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. The concentration and purity of the RNA samples were checked using a Nanodrop 2000 Biophotometer (Thermo Fisher Scientific Inc., West Palm Beach, FL, USA; pure RNA samples were indicated by a 260/280 nm absorbance ratio of 1.8–2.0) and electrophoresed to verify their integrity. Two-microgram RNA samples in a 20 μl reaction volume were reverse transcribed to cDNA using ImProm-IITM Reverse Transcriptase (Promega Biotech Co., Ltd., China).
Total protein was isolated from the liver, backfat and longissimus dorsi muscle using SDS Lysis Buffer (P0013B, Beyotime Ltd. China). Protein content was measured using an enhanced BCA protein assay kit (P0010, Beyotime, Ltd. China).
Total RNA was extracted from tissues with TRIZOL® Reagent (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instructions. The concentration and purity of the RNA samples were checked using a Nanodrop 2000 Biophotometer (Thermo Fisher Scientific Inc., West Palm Beach, FL, USA; pure RNA samples were indicated by a 260/280 nm absorbance ratio of 1.8–2.0) and electrophoresed to verify their integrity. Two-microgram RNA samples in a 20 μl reaction volume were reverse transcribed to cDNA using ImProm-IITM Reverse Transcriptase (Promega Biotech Co., Ltd., China).
Total protein was isolated from the liver, backfat and longissimus dorsi muscle using SDS Lysis Buffer (P0013B, Beyotime Ltd. China). Protein content was measured using an enhanced BCA protein assay kit (P0010, Beyotime, Ltd. China).
5
Transcriptome analysis using RNA-seq
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Total RNA was isolated using Trizol® reagent (Invitrogen, Waltham, MA, USA)55 (link). The integrity and concentration and purity of each sample was evaluated via 1% agarose gel electrophoresis and using a NanoDrop™ 2000 Biophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Samples were then reverse transcribed into cDNA using Superscript II reverse transcriptase (Invitrogen) and random hexamer primers. For RNA-seq analyses, RNA pools were created using equal quantities of RNA from four individuals; two biological replicates were included in each group. RNA-seq libraries were constructed according to the manuals provided by Illumina, Inc. (San Diego, CA, USA), and were sequenced using the HiSeq 2000 platform to generate 100-bp paired-end reads. All RNA sequencing data are deposited in the Gene Expression Omnibus under accession number GSE92981.
6
Multi-tissue Genomic and Molecular Analysis
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Genomic DNA was isolated from ear tissue as previously described [10 ], dissolved in TE solution, and stored at -20 °C.
Total RNA was extracted from the heart, liver, and kidney with TRIZOL® Reagent (Invitrogen, San Diego, CA, USA), checked for concentration and purity using a NanoDrop 2000 Biophotometer (Thermo Fisher Scientific Inc., West Palm Beach, FL, USA), and separated by electrophoresis in a 1 % agarose gel to verify integrity. After treatment with DNase I, 2 μg of RNA in a 20 μL reaction volume was reversely transcribed into cDNA using a SuperRT cDNA Kit (CWBIO Ltd., Beijing, China).
Total protein was isolated from the heart, liver, and kidney using SDS Lysis Buffer (P0013B, Beyotime Ltd., China). Protein content was measured with the enhanced BCA protein assay kit (P0010, Beyotime, Ltd., China).
Total RNA was extracted from the heart, liver, and kidney with TRIZOL® Reagent (Invitrogen, San Diego, CA, USA), checked for concentration and purity using a NanoDrop 2000 Biophotometer (Thermo Fisher Scientific Inc., West Palm Beach, FL, USA), and separated by electrophoresis in a 1 % agarose gel to verify integrity. After treatment with DNase I, 2 μg of RNA in a 20 μL reaction volume was reversely transcribed into cDNA using a SuperRT cDNA Kit (CWBIO Ltd., Beijing, China).
Total protein was isolated from the heart, liver, and kidney using SDS Lysis Buffer (P0013B, Beyotime Ltd., China). Protein content was measured with the enhanced BCA protein assay kit (P0010, Beyotime, Ltd., China).
7
RNA Extraction and Sequencing from Plant Leaves
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RNA was extracted from 100 mg of frozen leaf material powdered with liquid nitrogen using Trizol® reagent (Invitrogen, USA) and the PureLink® RNA Mini Kit (Ambion) according to the manufacturer’s protocol. Total RNA concentration and quality (260/280 nm = 2.0 ± 0.1) were checked with the NanoDrop® 2000 Biophotometer (Thermo Fisher Scientific, USA) and considered for sufficient purity. RNA integrity was verified with 1% agarose gel by electrophoresis and using the RNA 6000 Nano LabChip Kit and a Bioanalyzer 2100 (Agilent Technologies Inc., USA). Samples of RNA integrity number ≥ 6.5 were pooled to generate libraries with the TruSeq RNA Sample Preparation Kit, Set A (Illumina Inc., USA). Paired-end sequences (2 x 125 bp) were generated via the HiSeq 2500 (Illumina HiScanSQ platform) at the Centro de Genômica Funcional Aplicada a Agropecuária e Agroenergia, ESALQ, USP, Piracicaba, São Paulo, Brazil.
8
RNA Extraction and cDNA Synthesis Protocol
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Total RNA was isolated from lung tissue with Trizol Reagent (Invitrogen, Carlsbad, CA, USA) following manufacturer instructions. RNA solutions were evaluated for concentration and purity using a NanoDrop 2000 Biophotometer (Thermo scientific, Waltham, MA, USA) at 260/280 nm absorbance ratio and on a 1% agarose gel to verify DNA integrity.
Total RNA was reverse-transcribed into cDNA using FastQuant RT Kit (using gDNase, Tiangen, Beijing, China). Next, 1 µg total RNA and 2 µL 5X gDNA Buffer were added into a 200-µL microcentrifuge tube, and RNase-Free ddH 2 O was added to 10 µL volume. The tubes were incubated at 42°C for 3 min and then placed on ice. The reaction system contained 2 µL 10X Fast RT Buffer, 1 µL RT Enzyme Mix, and 2 µL FQ-RT Primer Mix. The samples were incubated at 42°C for 15 min, followed by incubation at 95°C for 3 min. The resulting cDNA was stored at 4°C for subsequent use.
Total RNA was reverse-transcribed into cDNA using FastQuant RT Kit (using gDNase, Tiangen, Beijing, China). Next, 1 µg total RNA and 2 µL 5X gDNA Buffer were added into a 200-µL microcentrifuge tube, and RNase-Free ddH 2 O was added to 10 µL volume. The tubes were incubated at 42°C for 3 min and then placed on ice. The reaction system contained 2 µL 10X Fast RT Buffer, 1 µL RT Enzyme Mix, and 2 µL FQ-RT Primer Mix. The samples were incubated at 42°C for 15 min, followed by incubation at 95°C for 3 min. The resulting cDNA was stored at 4°C for subsequent use.
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