Sybr green pcr master mix

RNA was isolated with RNeasy Plus Kit (QIAGEN, Valencia, CA) with on-column DNase treatment. 500 ng of RNA was transcribed into cDNA using Reverse Transcription System (Promega, Madison, WI) according to manufacturer's instructions. RT-qPCR was performed using an ABI Prism 7000 Sequence Detection System (Applied Biosystems) with SybrGreen PCR Master Mix (Quanta Biosciences). The following primer sets were used: GAPDH (5'-ACCCAGAAGACTGTGGATGG-3', 5'-CACATTGGGGGTAGGAACAC-3'); LANA (5'-CCTGGAAGTCCCACAGTGTT-3', 5'-AGACACAGGATGGGATGGAG-3'); c-Myc (5'-CAACGTCTTGGAACGTCAGA-3', 5'-TCGTCTGCTTGAATGGACAG-3'); VEGFA (5'-AGCACAGCAGATGTGAATGC-3', 5'-AATGCTTTCTCCGCTCTGAA-3'). In every run, melting curve analysis was performed to verify specificity of products as well as water and–RT controls. Data were analyzed using the ΔΔCT method as previously described [21 (link)]. Target gene expression was normalized to GAPDH by taking the difference between CT values for target genes and GAPDH (ΔCT value). These values were then calibrated to the control sample to give the ΔΔCT value. The fold target gene expression is given by the formula: 2^–ΔΔCT.

Total extracted RNA (1 μg) was used for cDNA synthesis with Oligo(dT)20 primers (Thermo Fisher Scientific, Grand Island, NY). The cDNA was added to an optimized quantitative reverse transcription-PCR (qRT-PCR) mixture that contained 1X SYBR Green PCR master mix (Quanta Biosciences, Gaithersburg, MD) and 500 nmol/L gene-specific primers. Assays were performed in triplicate on a CFX thermocycler (Bio-Rad, Hercules, CA). Specific gene plasmids for controls and Beta-2-microglobulin (β2MG) were used in all PCR analyses [26 (link)]. The value of qRT-PCR was normalized with Beta-2-microglobulin. The primers of RASSF8 and IL-6 (Integrated DNA Technologies, Inc., Coralville, Iowa) are listed in Supplementary Table 1.

Total RNA was extracted and purified using Trizol according to the manufacturer’s instructions (Life Technologies, Carlsbad, CA, USA). Complementary DNA (cDNA) was made using 1 μg of RNA and the qScript cDNA Synthesis Kit (Quanta BioSciences, Beverly, MA, USA). Quantitative real-time PCR (qPCR) was performed using the SYBR Green PCR Master Mix (Quanta BioSciences, Beverly, MA, USA) on a Bio-Rad real-time PCR machine. Relative gene expression was calculated using the 2^–∆∆Ct method and normalized to 18S or Actin RNA. The primers used in the study are shown in Table 2.

Antigen-specific T-cells were plated onto round 12-well plates (10⁶ cells/ well) and activated with 5x10⁵ irradiated (25 gray) APC and 5μg/ml of MOG p35-55 in the presence or absence of relevant peptides. Total RNA from cells was isolated 24 hours following activation using the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). 2μg aliquot of the total RNA was reverse transcribed into cDNA using Bio-RT (Bio-Lab, Jerusalem, Israel), dNTPs and random hexamer primers. qRT-PCR was performed on Step One Plus, ABI instrument (Applied Biosystems, Grand Island, NY, USA) using SYBR Green PCR Master Mix (Quanta BioSciences, Gaithersburg, MD, USA). The values for the specific genes were normalized to Rpl13a (mouse) as housekeeping controls and the data are described in arbitrary units. PCR reactions were performed in duplicate. The specific primers used for qRT-PCR are available on request.

Total RNA from cells was isolated using the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany). A concentration of 1 μg aliquot of the total RNA was reverse transcribed into cDNA using Bio-RT (Bio-Lab, Jerusalem, Israel), dNTPs and random hexamer primers. qRT-PCR was performed on Step One Plus, ABI instrument (Applied Biosystems, Grand Island, NY, USA) using SYBR Green PCR Master Mix (Quanta BioSciences, Gaithersburg, MD, USA). The values for the specific genes were normalized to HPRT housekeeping gene using the ΔΔCt method.
“qPCR analysis for mitochondrial DNA” has been published previously [13 (link)][14 (link)],[15 (link)],. Total DNA was isolated using Quick-gDNA MiniPrep (Zymo Research, Cat. No. D3007). Mitochondrial DNA (mtDNA) was quantified with SYBR Green PCR Master Mix on Step One Plus, ABI instrument (Applied Biosystems, Grand Island, NY, USA). The relative mtDNA copy number was calculated from the subtraction of mtDNA copies to nuclear DNA copies. The relative fold change was then calculated based on the ΔΔCt method. Primers used in this study are shown in Table S3.

mRNA expression was assessed via quantitative real-time PCR (qRT-PCR)^{72 (link)}. Approximately 400 fly heads per n were isolated. Total RNA was isolated using TRIZOL and was reverse transcribed via oligo (dT) primers and Superscript II reverse transcriptase (Invitrogen; Carlsbad, CA, USA). qRT-PCR was performed using an Applied Biosystems Fast 7500 system with SYBR Green PCR master mix (Quanta Biosciences; Beverly, MA, UAS) and run in triplicate. Each qRT-PCR experiment was performed with independent RNA isolations and cDNA syntheses, and normalized to actin5C. Primers used (forward/reverse) were as follows: Cp1, 5′- CTCATGTGACGCTGCCCAAATC-3′/5′- CCAGCACAGGCGCCCTC-3′; GA25021, 5′- GACAGCATTGATTCTTCCCCTCC-3′/5′- GTGTGCCATTCCTCCTGGATG-3′; actin5C, 5′- AGCGCGGTTACTCTTTCACCAC-3′/5′- GTGGCCATCTCCTGCTCAAAGT-3′ (Fisher Scientific, Hampton, NH, USA). Primers for Cp1 readily detected endogenous Cp1 from repo-Gal4/+ flies, but this level of expression was not significantly altered in repo-Gal4/UAS-GA25021 flies (see Supplementary Fig. 5). Additionally, primers for GA25021 detected a product in repo-Gal4/UAS-GA25021 flies, but this signal was not altered by expression of Cp1 RNAi HMS00725 (see Supplementary Fig. 5). These findings confirm that the Cp1 and GA25021 primers were specific for their intended products.