Gene specific primers

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Gene-specific primers are short DNA sequences designed to target and amplify specific regions of genetic material. They are used as essential components in various molecular biology techniques, such as polymerase chain reaction (PCR), to selectively replicate and study targeted genes or gene sequences.

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Specific primers (81 citations) TAQMAN gene specific primers (6 citations) FAM conjugated gene specific primers (4 citations) Rhesus-specific TaqMan gene expression primers (4 citations) FAM-labeled gene-specific probes and primers (3 citations) TaqMan Gene Expression Assays specific PCR primers sets (3 citations) TaqMan PCR Master Mix with gene-specific primers (3 citations) Universal TaqMan master mix and primer/probe sets specific for the genes (2 citations) Gene-specific oligonucleotide primers (2 citations) Mouse specific Taqman gene primers (2 citations)

57 protocols using gene specific primers

Gene Expression Analysis via qRT-PCR

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Cells were cultured and treated with different concentrations of Tan I for 24 or 48 hours. After cell harvested, total RNA was extracted using TRIzol reagent (Takara), according to the manufacturer's instructions. cDNA was synthesized from RNA with a cDNA reverse transcription kit (Takara). Real‐time PCR was performed in triplicate using gene‐specific primers (Thermo Fisher Scientific), on the Stratagene Mx3005P PCR system (Agilent Technologies). mRNA levels were normalized to β‐actin levels. The gene‐specific primers used are listed in Table 1 and were checked for specificity before use.

Wang W., Li J., Ding Z., Li Y., Wang J., Chen S, & Miao J. (2019). Tanshinone I inhibits the growth and metastasis of osteosarcoma via suppressing JAK/STAT3 signalling pathway. Journal of Cellular and Molecular Medicine, 23(9), 6454-6465.

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RT-qPCR gene expression analysis

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Total RNA was extracted using Trizol following the manufacturer’s instructions and qPCR reactions were done on a QuantStudio™ 7 Flex Real-Time PCR System using the Power SYBR green RNA-to-Ct RT-qPCR kit and gene specific primers (ThermoFisher). 50 ng of RNA template was added to the PCR mix. (ThermoFisher). Reaction conditions were 48 °C for 30 min, 95 °C for 10 min followed by 40 cycles (95 °C for 15 s, 60 °C for 60 s). Primers used are listed in Supplementary Table 18.

Flaherty E., Zhu S., Barretto N., Cheng E., Deans M.P., Fernando M., Schrode N., Francoeur N., Antoine A., Alganem K., Halpern M., Deikus G., Shah H., Fitzgerald M., Ladran I., Gochman P., Rapoport J., Tsankova N., Mccullumsmith R., Hoffman G.E., Sebra R., Fang G, & Brennand K.J. (2019). Neuronal impact of patient-specific aberrant NRXN1α splicing. Nature genetics, 51(12), 1679-1690.

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RT-qPCR gene expression analysis

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Arabidopsis Mutant Growth Conditions

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All the Arabidopsis (A. thaliana) plants were grown in soil (Sunshine #1 N&O Mix, Sun Gro Horticulture Canada Ltd., Canada) under normal conditions with 21°C and a 16-h photoperiod of ∼125 μE m⁻²s⁻¹. The Arabidopsis mutants used in this study were obtained from the ABRC stock center (Col-0 background), including ech (SAIL_163_E09) (Gendre et al., 2011 (link)), sid2-1 (Nawrath and Métraux, 1999 (link)), jar1-1 (Staswick et al., 2002 (link)), ein2-1 (Roman et al., 1995 (link)), pmr4-1 (Nishimura et al., 2003 (link)), and the transgenic line NahG (Lawton et al., 1995 ). The Arabidopsis transgenic lines tagged with fluorescent proteins include: pECH:ECH-YFP (Gendre et al., 2011 (link)), pPEN1:GFP-PEN1 (Collins et al., 2003 (link)), pPEN3:PEN3-GFP (Stein et al., 2006 (link)), 35S:PDF1.2a-GFP (Watanabe et al., 2013 (link)), 35S:PR1-GFP (Watanabe et al., 2013 (link)), 35S:secRFP (Faso et al., 2009 (link)), 35S:PMA-GFP (Lefebvre et al., 2004 (link)), and pUBQ:GFP-PMR4 (Kulich et al., 2018 (link)). The homozygosity of T-DNA insertion mutants was genotyped by PCR with both gene-specific and T-DNA border primers. The point mutation lines were genotyped by sequencing the PCR products after amplification with gene-specific primers (Thermo Fisher Scientific, Canada). The primers used in this study are listed in Supplemental Table S1.

Liu L., Qin L., Safdar L.B., Zhao C., Cheng X., Xie M., Zhang Y., Gao F., Bai Z., Huang J., Bhalerao R.P., Liu S, & Wei Y. (2022). The plant trans-Golgi network component ECHIDNA regulates defense, cell death, and endoplasmic reticulum stress. Plant Physiology, 191(1), 558-574.

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Transcriptome Analysis using Microarray

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RNA extraction and Affymetrix microarray chips (HG U133 Plus 2.0, Santa Clara, CA) were processed as previously described^{6 (link),18 (link)}. Affymetrix GeneChip Command Console (AGCC, 3.0 V) was used to scan images during data acquisition. Affymetrix CEL files (GEO accession GSE30174) containing raw intensity data were imported into Partek Genomics Suite 6.6 (Partek Inc., St. Louis, MO), log transformed, and normalized using the robust multi-array average (RMA) algorithm. Partek batch removal analysis of variance (ANOVA) was used to eliminate differences due to batch variation. ANOVA with false discovery rate (FDR) correction was used to identify differentially expressed genes (FDR <5%). The gene of interest was further confirmed with a TaqMan-based real-time quantitative PCR (RT-qPCR) using gene-specific primers (Thermo Fisher Scientific, Waltham, MA). Following genomic DNA elimination, a first-strand RNA-cDNA PCR template was generated from 150 ng of RNA using the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-qPCR was performed on a QuantStudio 6 Flex instrument (Thermo Fisher Scientific), and the gene of interest was normalized to GAPDH endogenous control.

Feng L.R., Fernández-Martínez J.L., Zaal K.J., deAndrés-Galiana E.J., Wolff B.S, & Saligan L.N. (2018). mGluR5 mediates post-radiotherapy fatigue development in cancer patients. Translational Psychiatry, 8, 110.

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Transcriptomic Analysis of Blood Samples

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A total of 2.5 ml of blood was collected from each subject in a RNA PAXGene tube (Qiagen, Frederick, MD). RNA extraction and Affymetrix microarray chips (HG U133 Plus 2.0, Santa Clara, CA) were processed as previously described (Saligan et al., 2013 ). Affymetric GeneChip Command Console (AGCC, 3.0V) was used to scan images during data acquisition. Affymetrix CEL files containing raw intensity data were imported into Partek Genomics Suite 6.6 (Partek Inc., St. Louis, MO), log transformed, and normalized using the robust multiarray average (RMA) algorithm. Because the chips were processed on different days, Partek batch removal analysis of variance (ANOVA) was used to eliminate differences due to batch variation. ANOVA with false discovery rate (FDR) correction was used to identify differentially expressed genes between groups (FDR < 5%). The gene of interest was further confirmed with a TaqMan-based real-time quantitative PCR (RT-qPCR) using gene-specific primers ((Thermo Fisher Scientific, Waltham, MA). Following genomic DNA elimination, first-strand RNA-cDNA PCR template was generated from 150 ng of RNA using the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RT-qPCR was performed on a QuantStudio 6 Flex instrument (Thermo Fisher Scientific) and gene of interest was normalized to GAPDH endogenous control.

Feng L.R., Suy S., Collins S.P, & Saligan L.N. (2017). The role of TRAIL in fatigue induced by repeated stress from radiotherapy. Journal of psychiatric research, 91, 130-138.

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Pharmacological Reagents for In Vivo Studies

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Drugs utilized in this study were obtained as follows – rTM (Asahi Kasei Pharma Co., Tokyo, Japan); and sCR1 (CDX-1135; Celldex Therapeutics, MA, USA); αCD47Ab (murine-specific: MIAP 301 [sc-12731]; porcine-specific: BRIC-126 [sc-59079]; Santa Cruz Biotechnology, TX, USA).
Other major reagents were obtained from the following sources – RNAlater solution (Ambion/Thermo Fisher Scientific, TX, USA); OCT media (Tissue-Tek, ProSciTech, Australia); Bond Polymer Refine Detection Kit (Leica Biosystems, Newcastle upon Tyne, UK); immunofluorescence mounting media (Dako/Agilent Technologies, CA, USA); rat anti-mouse Ly-6G/Ly-6C antibody (RB6-8C5) (Biolegend, CA, USA); rabbit anti-rat IgG (BA-4001) (Vector Laboratories, CA, USA); RNA extraction kit (Bioline/Meridian Life Science, TN, USA); gene-specific primers (Thermo Fisher Scientific, MA, USA); complement C3 antibody (Thermo Fisher Scientific);vC9 polyclonal antibodies (Abcam, Cambridge, UK); goat anti-rabbit Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific); TUNEL staining kit (In Situ Cell Death Detection Kit, TMR Red; Sigma-Aldrich/Merck, MO, USA); University of Wisconsin (UW) solution (Bridge to Life, SC, USA); creatinine (Merck, Darmstadt, Germany); and DHE (Thermo Fisher Scientific)

Hameed A.M., Lu D.B., Burns H., Byrne N., Chew Y.V., Julovi S., Ghimire K., Zanjani N.T., P’ng C.H., Meijles D., Dervish S., Matthews R., Miraziz R., O’Grady G., Yuen L., Pleass H.C., Rogers N.M, & Hawthorne W.J. (2020). Pharmacologic targeting of renal ischemia-reperfusion injury using a normothermic machine perfusion platform. Scientific Reports, 10, 6930.

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Transient Transfection of Mutant AP2S1 in HEK Cells

Cited 4 times

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Wild-type and mutant pBI-CMV4-AP2S1 expression constructs were generated, as described(12 (link)) and transiently transfected into human embryonic kidney (HEK)-293 cells stably expressing CaSR (HEK-CaSR)(7 (link),8 (link)) using Lipofectamine 2000 (Life Technologies). The bidirectional pBI-CMV4 cloning vector was used because it facilitated the co-expression of AP2σ and red fluorescent protein (RFP).(12 (link)) Site-directed mutagenesis was used to generate the mutant AP2S1 constructs using the Quikchange Lightning Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and gene-specific primers (Sigma-Aldrich), as described.(25 (link)) Cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM)-Glutamax media (ThermoFisher) with 10% fetal bovine serum (Gibco) and 400 μg/mL geneticin (ThermoFisher) at 37 °C, 5% CO₂. Transfection was confirmed by Western blot analyses and by visualizing RFP fluorescence using an Eclipse E400 fluorescence microscope with a Y-FL Epifluorescence attachment and a triband 4,6-diamidino-2-phenylindole-FITC-Rhodamine filter, and images taken using a DXM1200C digital camera and NIS Elements software (Nikon), as described.(12 (link))

Gorvin C.M., Rogers A., Stewart M., Paudyal A., Hough T.A., Teboul L., Wells S., Brown S.D., Cox R.D, & Thakker R.V. (2017). N‐ethyl‐N‐nitrosourea–Induced Adaptor Protein 2 Sigma Subunit 1 (Ap2s1) Mutations Establish Ap2s1 Loss‐of‐Function Mice. JBMR Plus, 1(1), 3-15.

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Cloning and Expression of M. tuberculosis rv3619c

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Genomic DNA isolated from M. tuberculosis H37Rv (obtained from the American Type Culture Collection, Rockville, MD, USA) served as the source for the amplification and subsequent cloning of the rv3619c gene, as previously described [9 (link)]. In brief, DNA corresponding to the rv3619c gene was amplified by PCR using genomic DNA isolated from M. tuberculosis and gene-specific primers (ThermoFisher Scientific, Ulm, Germany) (online suppl. Table S1; see www.karger.com/doi/10.1159/000525136 for all online suppl. material) and then ligated to appropriated cloning and expression vectors i.e., expression vector pGES-TH1 [9 (link)], shuttle vector pDE22 [12 (link)], and DNA plasmid vector pUMVC6 [12 (link)] (Aldevron, Fargo, ND, USA), as described previously [9 (link), 11 (link), 12 (link)] for expression in E. coli BL-21 (Novagen, Madison, WI, USA), M. smegmatis (ATCC 700084/mc(2)155, ATCC, Manassas, VA, USA), and DNA plasmid vector pUMVC6, respectively.

Safar H.A., Mustafa A.S., Amoudy H.A, & El-Hashim A. (2022). The Effect of Delivery Systems on the Induction of T Helper 1 Cell Response to an ESAT6-Like Protein Rv3619c and Identification of Its Immunodominant Peptides. Medical Principles and Practice, 31(4), 359-367.

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Vaginal Epithelial Cell Gene Expression

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Total RNA was extracted from vaginal epithelial cells from pre-differentiated (day 5 of expansion) and differentiated (day 10 of differentiation; exposed to 0.4 nM and 4 nM of β-estradiol for 10 days) vagina chips using QIAzol lysis reagent (Qiagen, cat. no. 79306). Complimentary DNA was synthesized using a SuperScript VILO MasterMix (Invitrogen, cat. no. 11755-500). The cellular gene-expression levels were determined using RT–qPCR, according to the TaqMan fast advanced master mix (ThermoFisher, cat. no. 4444964) with 20 μL of a reaction mixture containing gene-specific primers (ThermoFisher) for estrogen receptor (ESR1, Hs01046816), progesterone receptor (PGR, Hs01556702), phosphoenolpyruvate carboxykinase 1 (PCK1, Hs00159918), claudin 17 (CLDN17, Hs01043467), glucagon receptor (GCGR, Hs00164710), keratin15 (KRT15, Hs00951967), and zonula occludens-1 (ZO-1, Hs01551871). The expression levels of the target genes were normalized to GAPDH (Hs04420632).

Mahajan G., Doherty E., To T., Sutherland A., Grant J., Junaid A., Gulati A., LoGrande N., Izadifar Z., Timilsina S.S., Horváth V., Plebani R., France M., Hood-Pishchany I., Rakoff-Nahoum S., Kwon D.S., Goyal G., Prantil-Baun R., Ravel J, & Ingber D.E. (2022). Vaginal microbiome-host interactions modeled in a human vagina-on-a-chip. Microbiome, 10, 201.

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