Primer express software

Manufactured by Thermo Fisher Scientific

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Primer Express software is a computational tool designed for the design of real-time PCR primers and probes. It automates the process of selecting optimal oligonucleotide sequences based on user-specified criteria, such as melting temperature, GC content, and amplicon length.

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Primer Express (280 citations) Primer Express Software v3.0 (80 citations) Primer Express software version 3.0 (64 citations) Methyl Primer Express Software v1.0 (51 citations) Primer Express Software v2.0 (35 citations) Primer Express software, version 2.0 (27 citations) Primer Express software 3.0 (24 citations) Methyl Primer Express software (21 citations) Primer Express design software (6 citations) Primers Express software (5 citations)

675 protocols using primer express software

qRT-PCR Analysis of mRNA Levels

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mRNA isolation was carried out with TRIzol (Invitrogen) according to standard procedure. QRT-PCR was performed using SYBR^® Green I (Eurogentec, Seraing, Belgium) and Multiscribe Reverse Transcriptase (Applied Biosystems) and subsequently monitored by the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA). Primer sequences for each cDNA were designed using either Primer Express Software (Applied Biosystems) or qPrimer depot (http://primerdepot.nci.nih.gov) and are available upon request.
Detection of RPL0 gene was used to normalize the results. Primer sequences for each cDNA were designed using either Primer Express Software (Applied Biosystems) or qPrimer depot (http://primerdepot.nci.nih.gov), and these sequences are available upon request.

Habel N., El-Hachem N., Soysouvanh F., Hadhiri-Bzioueche H., Giuliano S., Nguyen S., Horák P., Gay A.S., Debayle D., Nottet N., Béranger G., Paillerets B.B., Bertolotto C, & Ballotti R. (2021). FBXO32 links ubiquitination to epigenetic reprograming of melanoma cells. Cell Death and Differentiation, 28(6), 1837-1848.

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QPCR-Based Gene Expression Analysis

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Transcript levels were determined by quantitative PCR (QPCR) using SYBR^® Green PCR Master Mix (Life Technologies, Rhenium, Modi’in, Israel). (https://www.sigmaaldrich.com/technical-documents/protocols/biology/sybr-green-qpcr.html). Gene specific primers were designed using the Primer Express Software (Life Technologies, Rhenium, Modi’in, Israel). The qPCR primer pairs were designed across exons to avoid false positive signals from potentially contaminating genomic DNA. Primer and cDNA concentrations were optimized (including melt curve analyses). The internal reference was ROX. Primer sequences are available upon request. Commercial software (Applied Biosystems, CA, USA) was used to calculate ΔΔCt (2^−(Ct_{Target gene}^{− Ct}_{control gene}) relative expression values for all the genes studied, normalized to control.

Ben-Dror K, & Birk R. (2019). Oleic acid ameliorates palmitic acid-induced ER stress and inflammation markers in naive and cerulein-treated exocrine pancreas cells. Bioscience Reports, 39(5), BSR20190054.

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Confirming Homozygous Deletions by ddPCR

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The homozygous deletion of CHST11 and MIR3922 identified by WGS of DNA from the proband’s cultured fibroblast cells was confirmed by testing the proband’s DNA from both her cultured fibroblasts and buccal epithelia simultaneously by ddPCR. TaqMan assays recognizing target genes were designed for a region on intron 1 (also overlapping MIR3922), exon 2 of CHST11, along with reference genes RPP30 and AP3B1 (sequences and other information can be found in Table S1). Each assay employed forward and reverse primers and an MGB-TaqMan probe labeled with either FAM- or VIC- dyes custom-designed using PrimerExpress software (Life Technologies, Waltham, MA) and synthesized by Life Technologies. Assays were designed specifically to exclude a MseI cutting site within the amplicon since MseI was used to fragment DNA into smaller pieces and to avoid copy number polymorphisms in the general population in the Database of Genomic Variants (MacDonald et al. 2013 (link)). The detailed workflow for ddPCR has been described previously (Hindson et al. 2011 (link)). Raw data analysis was automatically performed by QuantaSoft software (Bio-Rad, Hercules, CA), followed by manual normalization of the raw copy number values using a correction factor to yield a copy number of 2.0 to the RPP30 reference gene.

Chopra S.S., Leshchiner I., Duzkale H., McLaughlin H., Giovanni M., Zhang C., Stitziel N., Fingeroth J., Joyce R.M., Lebo M., Rehm H., Vuzman D., Maas R., Sunyaev S.R., Murray M, & Cassa C.A. (2015). Inherited CHST11/MIR3922 deletion is associated with a novel recessive syndrome presenting with skeletal malformation and malignant lymphoproliferative disease. Molecular Genetics & Genomic Medicine, 3(5), 413-423.

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Comprehensive RNA Extraction and RT-PCR

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Total RNA was prepared as described (8 (link), 11 (link), 12 (link)). For cDNA synthesis, 1 µg of total RNA was incubated for 90 min at 42°C in a total volume of 20 µl containing 50 mM Tris-HCl pH 8.3, 50 mM KCl, 4 mM MgCl₂, 10 mM DTT, 1 µM dT₁₈ primer, 1 mM dNTPs, and 200 U of RevertAid H Minus M-MuLV Reverse Transcriptase (Thermo Scientific, Braunschweig, Germany). RT-PCR for gene expression analysis was performed applying the ABI PRISM 7500 Sequence Detection System (Life Technologies, Darmstadt, Germany) as we described recently (11 (link)). Primers for HPRT, NKG2D were designed using Primer Express Software (Life Technologies) and validated. Primers for HLA-DRB1, CC-chemokine ligand 19 (CCL19), Leptin, DNAX accessory molecule 1 (DNAM-1), and MICA were purchased as Assays on Demand (Life Technologies) (Supplemental Table 1).

Resch T., Hackl H., Esser H., Günther J., Schwelberger H., Ritschl P.V., Ebner S., Maglione M., Mellitzer V., Biebl M., Öllinger R., Zoller H., Schneeberger S, & Kotsch K. (2021). Expression of MICA in Zero Hour Biopsies Predicts Graft Survival After Liver Transplantation. Frontiers in Immunology, 12, 606146.

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Isolation and Quantification of Mouse Kidney RNA

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Total RNA was isolated from snap‐frozen mouse kidney tissues using the RNeasy Mini Kit (Qiagen, Hilden, Germany), following the manufacturer's instructions. For cDNA synthesis, 2 µg RNA was reverse transcribed using oligo(dT) primer and RevertAid H Minus M‐MuLV Reverse Transcriptase (Fermentas GmbH, St. Leon‐Rot, Germany). Quantitative reverse transcription PCR was performed with the ABI PRISM 7500 Sequence Detection System using primers designed with Primer Express Software (Life Technologies, Darmstadt, Germany). The data were normalized to the housekeeping gene hypoxanthine‐guanine phosphoribosyltransferase (HPRT).

Ashraf M.I., Schwelberger H.G., Brendel K.A., Feurle J., Andrassy J., Kotsch K., Regele H., Pratschke J., Maier H.T, & Aigner F. (2015). Exogenous Lipocalin 2 Ameliorates Acute Rejection in a Mouse Model of Renal Transplantation. American Journal of Transplantation, 16(3), 808-820.

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Real-Time RT-PCR for Ovine Gene Expression

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About 500ng RNA from each tissue was reverse transcribed into cDNA using the SuperScript Vilo cDNA synthesis kit (Invitrogen). Gene expression was assessed by real time RT-PCR on a myiQ iCycler instrument (BioRad, Carlsbad, CA) using SYBRgreen based technique as described previously (Puttabyatappa, Andriessen et al. 2017 (link)). Sequences for oligonucleotide primers were based on previous publications or designed using Primer Express software (Life Technologies) and their sequences are shown in Supplemental Table 1. The relative fold change for each transcript was calculated using the ΔΔCT method following normalization to the endogenous reference gene ribosomal protein L19 (RPL19). Reference gene RPL19 was chosen because of its stable expression in the ovine tissues (Vorachek, Hugejiletu et al. 2013 (link), Jiang, Xue et al. 2015 (link)) and lack of effect of either prenatal T or bisphenol A treatment on its expression in ovine metabolic tissues used in this study (Supplemental Table 2).

Puttabyatappa M., Ciarelli J.N., Chatoff A.G, & Padmanabhan V. (2021). Developmental Programming: Metabolic Tissue-Specific Changes in Endoplasmic Reticulum Stress, Mitochondrial Oxidative and Telomere Length Status induced by Prenatal Testosterone Excess in the Female Sheep. Molecular and cellular endocrinology, 526, 111207.

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Quantitative RT-PCR Analysis of Host Cell Response

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TaqMan qRT-PCR was employed as described^{20 (link)} to assess the mRNA gene transcripts of MOMP (as a marker of C. trachomatis infectivity of macrophages), TLR2 (C. trachomatis cognate receptor) and the CD80 co-stimulatory molecule. TaqMan qRT-PCR was conducted using TaqMan RNA-to-Ct 1-step kit and TaqMan gene expression assay (for TLR2, CD80 and GAPDH) according to the manufacturer’s instructions (Life Technologies). MOMP primers and probe were designed using primer express software (Life Technologies). All values were normalized with respect to the GAPDH “housekeeping” gene mRNA levels. Results are presented as the fold increase over control (unstimulated cells) using the ΔΔCT method^{20 (link)}.

Dixit S., Singh S.R., Yilma A.N., Agee RD I.I., Taha M, & Dennis V.A. (2014). Poly (lactic acid)-poly (ethylene glycol) nanoparticles provide sustained delivery of a Chlamydia trachomatis recombinant MOMP peptide and potentiate systemic adaptive immune responses in mice. Nanomedicine : nanotechnology, biology, and medicine, 10(6), 1311-1321.

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Quantitative Analysis of Alcohol-Induced Gene Expression

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Total RNA from cultured cells was isolated using the RNeasy^® Plus Mini Kit and procedures provided by the manufacturer (Qiagen, Valencia, CA). RNA (12 ng) was subjected to 2-step real-time reverse transcription and polymerase chain reaction (RT-PCR) using iScript™ Reverse Transcription Supermix kit and SsoFast™ EvaGreen^® Supermix kit (Bio-Rad, Hercules, CA), respectively, on the CFX96™ Real-Time System (Bio-Rad). The amplification primers used for determinations are listed as follows:
These sets of primers for human cyclin D1, Snail, α-SMA, FSP1, and 18S rRNA were designed using Primer Express software (Life Technologies). The expression of cyclin D1, Snail, α-SMA, and FSP1 mRNA was determined by normalizing the cycle threshold (CT) number of their individual mRNA with that of 18S rRNA in each sample. Alcohol exposure induced changes of cyclin D1, Snail, α-SMA, and FSP1 mRNA expression are expressed as fold alterations over baseline expression by cells cultured simultaneously without exposure to alcohol.

Shi X., Chang C.C., Basson M.D., Upham B.L., Wei L, & Zhang P. (2014). Alcohol Disrupts Human Liver Stem/Progenitor Cell Proliferation and Differentiation. Journal of stem cell research & therapy, 4(5), 205.

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Quantitative real-time RT-PCR analysis

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Total RNA was prepared from mouse tissues using the RNA STAT-60 kit (TEL-TEST ‘B’, Friendswood, TX). Equal amounts of RNA from individual mice were treated with DNase I (DNA-free, Ambion/Life Technologies, Grand Island, NY). First strand cDNA was synthesized from 10 µg of DNase I-treated total RNA with random hexamer primers using TaqMan Reverse Transcription Reagents (Applied Biosystems/Roche, Branchburg, NJ). Specific primers for each gene were designed using Primer Express software (Life Technologies). The real-time RT-PCR reaction was set up in a final volume of 20 µl containing 20 ng of reverse-transcribed total RNA, 167 nM of the forward and reverse primers, and 10 µl of 2X SYBR Green PCR Master Mix (Life Technologies). The relative amount of all mRNAs was calculated using the comparative threshold cycle (C_T) method. Mouse cyclophilin mRNA was used as the invariant control.

Jo Y., Hamilton J.S., Hwang S., Garland K., Smith G.A., Su S., Fuentes I., Neelam S., Thompson B.M., McDonald J.G, & DeBose-Boyd R.A. (2019). Schnyder corneal dystrophy-associated UBIAD1 inhibits ER-associated degradation of HMG CoA reductase in mice. eLife, 8, e44396.

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Quantitative Real-Time PCR Protocol

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Primers and probes for the target β-actin gene and the internal standard were designed by using Primer Express Software, version 3.0 (Life Technologies, Carlsbad, CA, USA). The reverse primer for both the β-actin gene and internal standard was located in the 41-bp corresponding sequence. The sequences were as follows: (a) forward primers, 5'-GGACCTGACTGACTACCTCATGAA-3' (β-actin), 5'-AAACAGCTATGACCATGATTACGAA-3' (internal standard); (b) common reverse primer, 5'-CTTAAT GTCACGCACGATTTCC-3' (β-actin and internal standard); (c) probes, 5'-JOE-CACCGAGCGCGGCTACAGCTTC-ECLIPSE-3' (β-actin), 5'-FAM-CCACCAACCGCTCCCGTAATCTCTAG-ECLIPSE-3' (internal standard), JOE and FAM representing 2,7-dimethyl-4,5-dichlorine-6-carboxyfluorescein and 6-carboxyfluorescein, ECLIPSE, respectively, are kind of non-fluorescent quenchers. All PCR primers and fluorescent probes were custom-synthesized by Invitrogen (Shanghai, China).

Chen D., Pan S., Xie E., Gao L., Xu H., Xia W., Xu T, & Huang P. (2016). Development and Evaluation of a Duplex Real-Time PCR Assay With a Novel Internal Standard for Precise Quantification of Plasma DNA. Annals of Laboratory Medicine, 37(1), 18-27.

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