In this investigation, we carried out all experiments according to the experiment protocol guidelines (No. 2023030208) that had been reviewed and approved by the Experimental Animal Ethics and Welfare Committee of Shenyang Agricultural University (Shenyang, China). Six goats (adult and female) were sampled from the Liaoning cashmere breed without traceable genetic relationships. The skin tissue from each goat was collected according to the described assay by Zhu and colleagues [2 (link)]. In brief, the sampled goats were subjected to local anesthesia (posterior margin location of scapula), and the skin tissue (about 1 cm2) was collected using a sterilized surgical blade. After soaking and cleaning with 75% alcohol, the skin tissue was cut into pieces of 5 mm2. After washing three times using, the tissue samples were digested with 0.25% dispase II at 4 °C overnight. Under a stereo microscope, we separated the SHFs from skin tissue using a sterilized scalpel and disposable syringe needle. Finally, the SHF bulge section was separated according to the described assay by Ohyama and Kobayashi [17 (link)]. Using the RNAiso reagent kit, the total RNA was extracted according to the manufacturer’s instructions (TaKaRa, Dalian, China).
Rnaiso reagent kit
Manufactured by Takara Bio
Sourced in China
RNAiso reagent kit is a solution designed for the isolation and purification of total RNA from various biological samples. It utilizes a phenol-chloroform extraction method to effectively separate RNA from DNA and proteins.
Automatically generated - may contain errors
Lab products found in correlation
C 1 magnetic beads, by Thermo Fisher Scientific (2 mentions)
Dispase 2, by Roche (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Abi stepone real time pcr system, by Thermo Fisher Scientific (1 mentions)
One step primescript microrna cdna synthesis kit, by Takara Bio (1 mentions)
Primer premier 5.0 program, by Premier Biosoft (1 mentions)
Sybr green 1 assay, by Takara Bio (1 mentions)
M mulv cdna synthesis kit, by Sangon (1 mentions)
Prime script pt reagent kit, by Takara Bio (1 mentions)
Lightcycler 480 2 real time pcr system, by Roche (1 mentions)
Reverse transcription kit, by Takara Bio (1 mentions)
Lightcycler 480 2 real time pcr, by Roche (1 mentions)
Sybr premix ex taq, by Takara Bio (1 mentions)
Taqman microrna kit, by Thermo Fisher Scientific (1 mentions)
Miscript reverse transcription kit, by Qiagen (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
13 protocols using rnaiso reagent kit
1
Isolating Cashmere Goat Hair Follicle Stem Cells
2
Fungal Pathogenicity Genes Expression
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The gene expressions of fungal pathogenicity-linked genes as identified through genome-wide analysis and those involved in mycotoxins production were analyzed. The mycelia were harvested from F. graminearum grown in PDB medium and treated for 12 h with the cultures of Bacillus strains FZB42 and TS1 grown at 15 and 25 °C in LB medium. The mycelia treated with only LB medium served as control. Total RNA was extracted after grinding the mycelia in liquid nitrogen and using the Takara RNAiso Reagent Kit (Takara Bio, Beijing, China) by following the manufacturer’s guidelines. The cDNA synthesis and qPCR were performed similarly as described in Section 2.6 , with actin being used as the housekeeping gene. The qPCR primers for the expression analysis of the fungal pathogenicity-linked genes used in this study are listed in Table S2 .
3
RNA-seq for Liver Transcriptome
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Total liver RNA was isolated by using the RNAiso Reagent Kit (Takara, Dalian, China). RNA-sequencing of three biological replicates from each group was commissioned to BGI Tech (Shenzhen, China). In brief, the isolated RNA samples were treated with DNase I to remove DNA, and enriched by using oligo (dT) magnetic beads. The mRNA was fragmented into short fragments (about 200 bp), and converted to double strand cDNA. The cDNA was purified with magnetic beads, and sequencing adaptors were ligated to the fragments. The fragments were enriched by PCR amplification. The library products were sequenced on an Illumina HiSeq 2000 machine (BGI, Shenzhen, China).
4
Isolation and Extraction of Cashmere Goat Hair Follicle Bulge
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In this study, all experimental protocols were reviewed and approved by the Experimental Animal Ethics and Welfare Committee of Shenyang Agricultural University (Shenyang, China) under the ethical code: 201606005. Correspondingly, the experiments were performed based on the approved procedure guidelines. The skin tissues (body side) were collected from nine cashmere goats (Liaoning cashmere breed, female, three-year-old, no traceable common genetic relationships) as described in our previous investigation [14 (link)]. In brief, the skin tissues were sampled from each goat using a sterilized surgical blade. The collected skin samples were cleaned with 75% alcohol. Subsequently, the skin samples were cut into pieces of 5 mm2 and washed with PBS for 3 times. The samples were further digested at 4°C with dispase II (0.25%; Roche, Mannheim, Germany) for 8 h. Under a stereo microscope, the SHFs were isolated with a sterilized scalpel and disposable syringe needle, and the SHF bulge sections were isolated following described protocol by Ohyama and Kobayashi [18 (link)]. The total RNA was extracted from SHF bulge using RNAiso reagent kit (TaKaRa, Dalian, China) according to the manufacturer’s recommendations.
5
Validation of lncRNAs and mRNAs in Chinese Cabbage
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For validation of the lncRNAs and mRNAs in Chinese cabbage, total RNA from each sample was extracted using the RNAiso Reagent Kit (TaKara, China). Single-strand cDNA was synthesized with approximately 2 μg RNA from each sample by using the RevertAid First Strand cDNA Synthesis Kit (Fermentas, USA). Using SYBR Premix Ex Taq Kit (TaKaRa, China), the qRT-PCRs were performed on an ABI StepOne Real-Time PCR System (Applied Biosystems, USA). The melting curve was used to verify only one specific product. Three replicates were performed for each sample. BrActin was used as the internal gene for mRNAs and lncRNAs. All the primers used in the study are listed in Table S11 . The relative expression levels of mRNAs and lncRNAs were normalized using a △△CT method74 (link). Significance of the expression level between the sensitive variety ‘GHA’and tolerant variety ‘XK’ were determined by one-way of variance with t-rest (P < 0.05 or P < 0.01).
6
Goat SHF Bulge RNA Profiling
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From goat SHF bulges and stem cells, the total RNA was isolated with the RNAiso reagent kit (TaKaRa, Dalian, China). Based on the use of random primers, the first strand cDNA was reversely transcribed by M-MuLV cDNA Synthesis Kit (Sangon, Shanghai, China). One Step PrimeScript microRNA cDNA synthesis kit (TaKaRa, Dalian, China) was used to transcribe the cDNA for the microRNAs testing. We carried out real-time PCR amplification using SYBR Green I assay (TaKaRa, Dalian, China). All primers were designed through the use of Premier Primer 5.0 program (Premier Biosoft International, Palo Alto, CA, USA). The analyzed miRNA mature sequences were obtained from the miRNA database (http://www.mirbase.org , accessed on 28 July 2019), and their sense primers were designed based on the the corresponding miRNA sequence. Whereas, the corresponding anti-sense primers were provided in the kits (TaKaRa, Dalian, China), which are universal reverse primers for all miRNAs analyses. Here, all primers are listed in Supplementary Table S1 with their detailed information. The PCR reaction of each sample was performed in triplicate.
7
Quantitative Real-Time PCR Gene Expression Analysis
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Total RNA extraction was performed using the RNAiso Reagent kit (Takara, China), and cDNA was synthesized using the Prime Script PT reagent Kit (Takara, China). Table 2 lists the forward and reverse primers of genes. qRT-PCR was performed using a LightCycler® 480 II Real-Time PCR system (Roche, Switzerland). The PCR temperature conditions were 95°C for 5 min, followed by 40 cycles of 95°C for 20 s, 57°C for 25 s, and 72°C for 25 s. Each sample was analyzed in triplicate, and the internal control genes included β-actin and GAPDH. The expression levels were calculated using the 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)).
8
RNA Extraction and Reverse Transcription
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The liver samples were pulverized in liquid nitrogen and transferred to a 1.5-ml RNAse-free Eppendorf tube. Total RNA was extracted using the RNAiso Reagent Kit (TaKaRa Corporation) according to the manufacturer's instructions. The extracted total RNA was examined for integrity by gel electrophoresis and for purity by determining absorbance (OD value) at 260 and 280 nm. Reverse transcription was performed using the Reverse Transcription Kit (TaKaRa Corporation), and the reverse transcription products were stored at −80°C for later use.
9
Isolation of Cashmere Goat Hair Follicles
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Skin tissue from the side body were collected from three healthy cashmere goats (belonging to the Liaoning cashmere goat breed, that were three years old, female, and had no common traceable genetic relationships) at anagen, catagen, and telogen using sterile scalpel blades. The SHFs were further isolated from the harvested skin tissues as described in previous publications [27 (link),28 (link)]. The total RNA was isolated from each sample using the RNAiso reagent kit (TaKaRa, Dalian, China) following the manufacturer’s instructions. The integrity of isolated total RNA was verified by agarose gel (1.5%) electrophoresis. Also, the purity and quantity of isolated total RNA were assessed through an ultraviolet spectrophotometer (Hoefer, San Francisco, CA, USA). For the ratio of OD260/OD280, all total RNA samples were verified to range from 1.8 to 2.0. Subsequently, the obtained total RNA was stored at −80 °C prior to further analysis.
10
Quantifying Gene Expression in Yellow Catfish
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Total RNA was extracted from the muscle tissue of yellow catfish using an RNAiso reagent kit (Takara Bio. Inc., China). Roche LightCycler®480 II real-time PCR instrument (Roche Ltd., Switzerland) and SYBR Premix Ex Taq (Takara Bio. Inc., China) were used for qPCR detection. Primer 6 was used to design specific primers for q-PCR (Table 2 ). The PCR temperature conditions were 95°C for 30 s followed by 40 cycles of 95°C for 20 s, 57°C for 25 s, and 72°C for 25 s. The expression levels were calculated using the 2−ΔΔCT method (28 (link)).
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