To extract the miRNAs from each sample, a high purity miRNA isolation Kit (Roche) was used. The solutions were quantified using the Qubit® 2.0 Fluorometer (Invitrogen) and diluted to a final concentration of 4 ng/μL. Then, the cDNA was obtained using TaqMan® MicroRNA Reverse Transcription Kit (Life Technologies), and the Quantitative Real-Time PCR reaction was performed using the TaqMan® MicroRNA Assays in a 7500 Real Time PCR system (Life Technologies) with TaqMan miRNA assays according to the manufacturer’s instructions (Life Technologies) using primers designed by Primer Express (Life Technologies). The mean expression level of three human endogenous controls (Z30, RNU19 and RNU6B – calibrators) was used as an internal control in all of the miRNA experiments to allow the comparison of expression results. The relative miRNA expression levels were then calculated by a comparative threshold cycle (Ct) method (2−Δct).
Primer express
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Italy, Germany, France
Primer Express is a software tool developed by Thermo Fisher Scientific for the design and selection of primers and probes for real-time PCR (polymerase chain reaction) experiments. The software's core function is to assist researchers in the identification of optimal primer and probe sequences that meet specific criteria for use in real-time PCR applications.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (37 mentions)
Rneasy mini kit, by Qiagen (24 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (18 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (15 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (14 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (12 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (9 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (9 mentions)
Sybr premix ex taq, by Takara Bio (9 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (8 mentions)
Trizol, by Thermo Fisher Scientific (7 mentions)
Nanodrop, by Thermo Fisher Scientific (7 mentions)
Tri reagent, by Molecular Research Center (6 mentions)
Primescript rt reagent kit, by Takara Bio (6 mentions)
Steponeplus, by Thermo Fisher Scientific (5 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Opticon 4 machine, by Bio-Rad (5 mentions)
Superscript reverse transcription system, by Takara Bio (5 mentions)
Abi prism 7000, by Thermo Fisher Scientific (5 mentions)
Lightcycler 480, by Roche (5 mentions)
280 protocols using primer express
1
Quantitative Analysis of miRNA
2
qRT-PCR Primer Design and Optimization
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The qRT-PCR primers for the genes of interest (Table S4 ) were designed by Primer Express, version 3.0 (Life Technologies, Carlsbad, CA, USA) to amplify approximately 100 bp segments from regions with uniform coverage in the RNA-Seq reads as confirmed using Geneious software. For primer optimization (90–110% efficiency) and qRT-PCR (Applied Biosystems 7300 Real-Time PCR System), the primers were all at 0.4 µM concentration, with the exception of CKS_3793 (0.6 µM), as this was optimized from a previous study (Kernell Burke et al., 2015 (link)). RNA for each sample type was harvested using the same methods as for the RNA-Seq following the miRNeasy kit protocol. Each sample had a RIN value of at least 7. Once extracted, the RNA was converted to cDNA using the ABI High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA). The Pfaffl method was used to determine the fold change differences between samples from the in planta culture and either the pre-inoculum in vitro liquid culture or in vitro plate culture.
3
KASP Assay for Soybean Rust Resistance
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KASP primer sequences were designed using PrimerExpress (Life Technologies, Carlsbad, CA), and are listed in S1 Table . KASP reactions were 4 μL in volume, with 2 μL of 2X premade KASP master mix (LGC, Middlesex, UK), 0.055 μL of primers mix (Sigma-Aldrich, St. Louis, USA), and 10–40 ng of genomic DNA. After PCR amplification, PCR plates were scanned with a Tecan M1000 Pro Infinite Reader (Tecan Group Ltd., Männedorf, Switzerland). The SNP genotype was determined using Kluster Caller software (LGC Genomics, Middlesex, UK).
To test the association between the SNPs identified in the two candidate genes Glyma13g25340 and Glyma13g25350 and the FLS phenotypes, and to develop robust SNP assays for genotyping and marker-assisted selection, one SNP specific for both PI 594891 and PI 594774 from each of the two candidate genes was selected for KASP assay development, This resulted in a total of four KASP assays. A set of 36 F2 seed from either Blackhawk x PI 594891 or Blackhawk x PI 594774 and a panel of 158 cultivars and germplasm lines were genotyped using these KASP assays. The 158 soybean lines in the panel were selected due to the availability of DNA for the genotyping experiment and they were from the list used in a previous experiment in our lab. Due to the limited availability of seeds, only 45 lines were assayed for their reactions to C. sojina.
To test the association between the SNPs identified in the two candidate genes Glyma13g25340 and Glyma13g25350 and the FLS phenotypes, and to develop robust SNP assays for genotyping and marker-assisted selection, one SNP specific for both PI 594891 and PI 594774 from each of the two candidate genes was selected for KASP assay development, This resulted in a total of four KASP assays. A set of 36 F2 seed from either Blackhawk x PI 594891 or Blackhawk x PI 594774 and a panel of 158 cultivars and germplasm lines were genotyped using these KASP assays. The 158 soybean lines in the panel were selected due to the availability of DNA for the genotyping experiment and they were from the list used in a previous experiment in our lab. Due to the limited availability of seeds, only 45 lines were assayed for their reactions to C. sojina.
4
Quantitative Real-Time PCR for BRCA1/2 Expression
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Primers and probes for the TATA box-binding protein (TBP;
endogenous RNA-control) were used as previously
described.13 (link) Primers and probes for BRCA2 [GenBank: NM_000059.3] were determined with the assistance of
the computer program Primer Express (Life Technologies, Carlsbad, CA, USA).
BRCA2 forward primer: 5′-GAA AAT CAA GAA AAA
TCC TTA AAG GCT-3′; BRCA2 reverse-primer:
5′-GTA ATC GGC TCT AAA GAA ACA TGA TG-3′; BRCA2TaqMan probe: 5′-FAM-AGC ACT CCA GAT GGC ACA ATA AAA GAT CGA AG-3′-TAMRA. Primers
and probe for BRCA1 were purchased from Applied
Biosystems (Foster City, CA, USA, Applied Biosystems Assay ID: Hs01556193_m1). PCR
reactions were performed as previously described.13 (link) Each experiment included a
standard curve with five cDNA concentrations, a positive control sample (OVCAR-3
carcinoma cell-line), 40 patient samples and a no template control. The standard
curves were generated using serially diluted solutions of standard cDNA derived
from the HTB-77 carcinoma cell line. The target mRNA quantity in each sample was
determined from the relative standard curve, data normalization was carried out
against TBP, the endogenous RNA-control and expressed in arbitrary units
corresponding to the dilution factors of the standard RNA preparation. Real-time
PCR assays were conducted in duplicates for each sample, and the mean value was
used for calculation.
endogenous RNA-control) were used as previously
described.13 (link) Primers and probes for BRCA2 [GenBank: NM_000059.3] were determined with the assistance of
the computer program Primer Express (Life Technologies, Carlsbad, CA, USA).
BRCA2 forward primer: 5′-GAA AAT CAA GAA AAA
TCC TTA AAG GCT-3′; BRCA2 reverse-primer:
5′-GTA ATC GGC TCT AAA GAA ACA TGA TG-3′; BRCA2TaqMan probe: 5′-FAM-AGC ACT CCA GAT GGC ACA ATA AAA GAT CGA AG-3′-TAMRA. Primers
and probe for BRCA1 were purchased from Applied
Biosystems (Foster City, CA, USA, Applied Biosystems Assay ID: Hs01556193_m1). PCR
reactions were performed as previously described.13 (link) Each experiment included a
standard curve with five cDNA concentrations, a positive control sample (OVCAR-3
carcinoma cell-line), 40 patient samples and a no template control. The standard
curves were generated using serially diluted solutions of standard cDNA derived
from the HTB-77 carcinoma cell line. The target mRNA quantity in each sample was
determined from the relative standard curve, data normalization was carried out
against TBP, the endogenous RNA-control and expressed in arbitrary units
corresponding to the dilution factors of the standard RNA preparation. Real-time
PCR assays were conducted in duplicates for each sample, and the mean value was
used for calculation.
5
Primer Design for Inflammatory Genes
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Primers and probes for TNFA, CYLD and RNF31 were purchased from Applied Biosystems (Hs00174128_m1, Hs01031576_m1, Hs00215938_m1). Primers and probes for SPATA2 (GenBank no. NM_001135773.1 ) were determined with the assistance of the computer program Primer Express (Life Technologies, Carlsbad, CA, USA): SPATA2 forward primer, 5′‐CCG TGG AAG AAG GAA TTC AGA A‐3′; SPATA2 reverse primer, 5′‐CCA GTA ATG TCG ACT TGA CAT AAT AAA CA‐3′; and SPATA2 TaqMan probe, 5′‐FAM‐CAT CAA GAC CTA CAC GGG CCC TT‐3′‐TAMRA. TBP was used as the reference gene. PCR reactions were performed as previously described.23
6
Quantifying gene expression in skeletal muscle
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RNA from skeletal muscle was extracted into TRizol reagent (Invitrogen 15596026) and reverse transcribed using a High-Capacity cDNA Reverse Transcription kit (Applied Biosystems 4368814), both following the manufacturer's recommendations. qRT-PCR was performed using SYBR Select Master Mix (Applied Biosystems 4472908) with primers designed in-house using Primer Express® (Table S1 ) on a ViiA®7 Real-Time PCR system in triplicate (Life Technologies). Quantification was performed using the 2−ΔCT method and normalised to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
7
Ultra PCR for HIV-1 and HCV Detection
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Example 1
A nucleotide acid sequence alignment was set up that contained HIV-1 sequences present in GenBank and the Los Alamos National Laboratory database (HIV-1 2012). Primers and probe binding in conserved domains were designed by analyses of sequences including alignment and further justified by the Primer Express software (Life Technologies) and related salted and unsalted buffer conditions. The primers and probes in Table 1 were designed for the detection of all subtypes of HIV-1 M group and most of 0 group, while primers and probes in Table 2 were designed for all HCV subtypes. Three pairs of primers were designed for triple rounds of PCR for each Ultra PCR. The first round PCR is designed for high specificity with primer pair designed with higher annealing temperature (65-71° C.). The second round PCR remains more specificity with primers at relatively high annealing temperature (60-64° C.) and increased amplification efficiency. The third round PCR is designed for the maximal amplification of the first and second rounds of PCR products with primers at low annealing temperature (50-55° C.). So, the combination of the first, second and third rounds of PCR significantly improve the sensitivity and specificity of Ultra PCR (see
8
Real-time PCR for SMN1 Detection
9
Endogenous RNA Control and L1CAM Quantification
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Primers and probes for the TATA box-binding protein (TBP; a component of the DNA-binding protein complex TFIID as an endogenous RNA control) were used according to Bieche et al. [34 (link)] Primers and probes for L1CAM were determined with the assistance of the computer program Primer Express (Life Technologies, Carlsbad, CA, USA). BLASTN searches were conducted to confirm the total gene specificity of the nucleotide sequences chosen for the primers and probes. To prevent amplification of contaminating genomic DNA, the probe was placed at the junction between exon 12 and exon 13. L1CAM forward primer: 5′-TTC GTC CTG AAG CAC TGT TGT C-3′; L1CAM reverse-primer: 5′-GGA GCG CCT GTG CCC-3′; L1CAM TaqMan probe: 5′-FAM-ATC CTC GTC CAG CCA CTG AAC A-3′-TAM.
10
Primer Design for Anguilla anguilla
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Candidate reference gene products as well as target transcripts and their functions are listed in Table 1 . Primers were obtained from previous studies or were designed with Primer Express (Life Technologies) (Table 2 ), using nucleotide sequences retrieved from the GeneBank database (https://www.ncbi.nlm.nih.gov/genbank/ ) for Anguilla anguilla or from Next Generation RNA sequencing (454 FLX Titanium) of A. anguilla transcriptome (EeelBase database16 (link)). Primer pair specificities were checked both in silico and empirically by BLAST analysis and using melting profiles. BLAST analyses against A. anguilla nucleotide sequences in the GenBank indicated all primers were specific (data not shown), which was confirmed by melting profiles, electrophoresis, and sequencing of PCR products.
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