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.
Primer premier 5.0 program
Manufactured by Premier Biosoft
Sourced in United States
Primer Premier 5.0 is a software program for designing and analyzing primers and probes for PCR (Polymerase Chain Reaction) applications. The program provides tools for primer and probe selection, analysis of melting temperatures, and evaluation of potential cross-reactivity.
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Lab products found in correlation
One step primescript microrna cdna synthesis kit, by Takara Bio (2 mentions)
M mulv cdna synthesis kit, by Sangon (2 mentions)
Sybr green 1 assay, by Takara Bio (1 mentions)
Rnaiso reagent kit, by Takara Bio (1 mentions)
Rnaiso kit, by Takara Bio (1 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions)
Trizol reagent, by Takara Bio (1 mentions)
One step gdna removal and cdna synthesis kit, by Transgene (1 mentions)
Sybr premix ex taq 2 tli rnaseh plus master mix, by Takara Bio (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Magnesphere paramagnetic particles, by Promega (1 mentions)
7 protocols using primer premier 5.0 program
1
Goat SHF Bulge RNA Profiling
2
Cashmere Goat Bulge circRNA-0100 Expression
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Here, RNAiso kits (TaKaRa, Dalian, China) were utilized for extracting
the total RNA from SHF bulges of cashmere goats and its stem cells. For the
expression detection of circRNA-0100 and related gene mRNAs, random primers
were utilized to synthesize the first strand cDNA with an M-MuLV cDNA synthesis
kit (Sangon, Shanghai, China), whereas for the expression detection of
miRNAs, the first strand cDNA was synthesized by a One-Step PrimeScript
microRNA cDNA synthesis kit (TaKaRa, Dalian, China). The divergent primers
for detecting the expression of circRNA-0100 were designed using the
CircPrimer program (Zhong et al., 2018). The convergent primers for mRNA
detection were designed by the Premier Primer 5.0 program (Premier Biosoft
International, Palo Alto, CA, USA). A combined internal control consisting
of UBC, YWHAZ, and SDHA was utilized for normalizing the gene expression
level (Bai et al., 2014). The corresponding mature sequences of all detected
miRNAs were retrieved from the miRNAsong database
(https://www2.med.muni.cz/histology/miRNAsong/index.php , last access: 16 April 2021). A combined
internal control consisting of let-7d-5p, miR-26a-5p, and miR-15a-5p was
utilized for normalizing the miRNA expression level (Bai et al., 2016). All
primers are provided in Table 1 with the corresponding detailed
information.
the total RNA from SHF bulges of cashmere goats and its stem cells. For the
expression detection of circRNA-0100 and related gene mRNAs, random primers
were utilized to synthesize the first strand cDNA with an M-MuLV cDNA synthesis
kit (Sangon, Shanghai, China), whereas for the expression detection of
miRNAs, the first strand cDNA was synthesized by a One-Step PrimeScript
microRNA cDNA synthesis kit (TaKaRa, Dalian, China). The divergent primers
for detecting the expression of circRNA-0100 were designed using the
CircPrimer program (Zhong et al., 2018). The convergent primers for mRNA
detection were designed by the Premier Primer 5.0 program (Premier Biosoft
International, Palo Alto, CA, USA). A combined internal control consisting
of UBC, YWHAZ, and SDHA was utilized for normalizing the gene expression
level (Bai et al., 2014). The corresponding mature sequences of all detected
miRNAs were retrieved from the miRNAsong database
(
internal control consisting of let-7d-5p, miR-26a-5p, and miR-15a-5p was
utilized for normalizing the miRNA expression level (Bai et al., 2016). All
primers are provided in Table 1 with the corresponding detailed
information.
3
Quantifying Expression of ncRNAs in Glioma
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Quantitative real‐time PCR (qRT‐PCR) was used to examine the expression of LOC441179, PON2 and USP46‐AS1 in glioma cells. Total RNAs were extracted from the glioma cells and control cells with TRIzol reagent (TaKaRa, Dalian, China) according to the manufacturer's instructions. cDNA synthesis was performed with PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa) according to the manufacturer's instructions. The primers specific of each ncRNA were designed using the primer premier 5.0 program (PREMIER Biosoft, Palo Alto, CA, USA) according to the sequences obtained from NCBI (Table 1 ). The β‐actin gene was chosen as the reference for internal standardization. The qRT‐PCR amplification was performed on an ABI 7500 real‐time PCR system (Applied Biosystems, Waltham, MA, USA) at 95 °C for 10 s, followed by 40 cycles of 95 °C for 5 s, 60 °C for 15 s, 72 °C for 35 s. Reaction of each sample was performed in triplicate. At the end of each reaction, dissociation analysis was performed to confirm the amplification specificity. The expression levels of LOC441179, PON2 and USP46‐AS1 relative to that of the β‐actin gene in glioma cells and control cells were calculated by the comparative CT method (2 − Δ Δ C T
4
Intron-Flanking Primers Design for P. ginseng ESTs
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50 EST sequences involved in three gene ontology categories (molecular function, biological process, and cellular component) were randomly selected from an EST database of P. ginseng [15 (link)]. Positions of introns were predicted by using the potential intron polymorphism (PIP) database [16 (link)]. EST sequences for which the corresponding genomic DNA sequences may contain introns were targeted for intron-flanking primers design. For the ESTs with available P. ginseng genomic DNA sequences, primers were designed according to the genomic DNA sequence. Primers flanking introns were designed by using Primer Premier 5.0 program (Premier Biosoft, Palo Alto, CA, USA).
5
Quantitative Analysis of Chemosensory Genes
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Antennae, proboscises, maxillary palps, thoraxes, legs, abdomens, heads (without antennae, proboscises, and maxillary palps), and wings (50 pairs of each sex) were collected for total RNA extraction using Trizol reagent (Invitrogen, Carlsbad, CA, USA). The first strand cDNA template was synthetized with One-Step gDNA removal and cDNA Synthesis kit (TransGen, Beijing, China) including oligo dt-primer according to product manual recommendations. The primers of CpunPBP2, CpunPBP5 and reference gene (β-actin, accession number JX119014 ) for real-quantitative PCR (qPCR) were designed using Primer premier 5.0 program (Premier Biosoft International, Palo Alto, CA, USA; Table S1 ). qPCR were conducted on ABI 7500 fast real-time PCR system (Applied Biosysterm, USA). Each amplification reaction was performed with 20 μL volume using SYBR Premix Ex Taq II (Tli RNaseH Plus) master mix (Takara-Bio, Shiga, Japan) under the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 3 s and 60°C for 30 s. To check reproducibility, each test sample was done in triplicate technical replicates and three biological replicates. Relative quantification was analyzed using the comparative 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The relative expression levels in different tissues were calculated with the transcript level of the female antennae used as the calibrator.
6
Multilocus Sequence Typing of Leuc. mesenteroides
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With reference to the genome sequence of Leuc. mesenteroides ATCC8293 (GenBank accession number: CP000414.1), 9 housekeeping genes (dnaA, groEL, murC, murE, pepN, pheS, pyrG, rpoB, and uvrC) were selected for MLST analysis. These genes are evenly separated across the entire genome, conserved, and well characterized (Cai et al., 2007; Bilhère et al., 2009) . The primers for these 9 genes were designed using the Primer Premier 5.0 program (Premier Biosoft International, Palo Alto, CA) and are listed in Table 1.
7
Microsatellite Marker Development for Pseudomonas aeruginosa
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Microsatellite loci were isolated from an enriched library according to the hybridization-based capture method described by Zane et al. (2002) and recommended by Gu et al. (2012a,b) . High-quality genomic DNA from individual B. aeruginosa was fragmented using the restriction enzyme MseI (BioLabs, USA), and then double-stranded DNA linkers specifically designed for this application (5'-GACGATGAGTCCTGAG-3' and 5'-TACTCAGGACTCAT-3') were added into the ends. The PCR products were size-selected to preferentially obtain small fragments (300-800 bp), which were subsequently hybridized to a mixture of 5'-biotinylated oligoprobes: (TC) 20 , (CAG) 15 , (ATG) 15 , (CCT) 8 , and (GATA) 8 . The oligoprobes were then captured by streptavidin MagneSphere paramagnetic particles (Promega). The enriched DNA fragments were amplified, cloned and sequenced. Finally, SSRs were screened using the SSRHUNTER program (Li and Wan, 2005) . A minimum repeat length criterion of 20 bp was used for selection. Primers flanking SSRs were designed using the PRIMER PREMIER 5.0 program (PREMIER Biosoft International, USA).
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