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Sybr green detection

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SYBR green is a nucleic acid stain used for the detection and quantification of double-stranded DNA. It binds to the minor groove of DNA, emitting a fluorescent signal upon excitation. This property allows for the real-time monitoring of DNA amplification during polymerase chain reaction (PCR) experiments.

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Lab products found in correlation

Rna clean concentrator kit, by Zymo Research (3 mentions) Tri reagent, by Merck Group (3 mentions) Ds 11 series spectrophotometer fluorometer, by DeNovix (3 mentions) Sybr primescript reverse transcription pcr rt pcr kit 2, by Takara Bio (2 mentions) Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions) Stratagene mx3000p qpcr, by Agilent Technologies (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions)

4 protocols using sybr green detection

1

Profiling Tick ATG Gene Expression

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Total RNA was extracted from cells using TRI Reagent (Sigma, St. Louis, MO, USA), and purified by an RNA Clean & Concentrator kit (Zymo Research, Irvine, USA). The quantity and quality of RNA were assessed via a DS-11 Series Spectrophotometer/Fluorometer (DeNovix, Wilmington, USA). Genomic DNA contamination was eliminated and cDNA was synthesized using PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Otsu, Shiga, Japan). The expression profile of tick ATG (17 (link)) at different infection rates was assessed using qPCR on the Mx3005P Real-Time system (Stratagene, La Jolla, USA) with SYBR green detection (Agilent Technologies, Santa Clara, USA). All protocols were according to the manufacturer’s instructions. Primers used in this study are listed in Table S1. The analysis of the Atg gene expression profile included a one-way analysis of variance for each gene, with subsequent Bonferroni correction to address multiple comparisons, and the glyceraldehyde 3-phosphate dehydrogenase (gapdh) gene from the I. scapularis tick genome as a reference control. Relative gene expression levels of individual Atg genes were calculated using the comparative cycle threshold (CT) method (2−ΔΔCT). Statistical analysis was performed using the data obtained from three separate cDNA sets from three independent biological samples.
Wang X.R., Cull B., Oliver J.D., Kurtti T.J, & Munderloh U.G. (2023). The role of autophagy in tick-endosymbiont interactions: insights from Ixodes scapularis and Rickettsia buchneri. Microbiology Spectrum, 12(1), e01086-23.
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2

Quantifying Autophagic Gene Expression in Ticks

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Total RNA was extracted from above samples using TRI Reagent® (Sigma, T9424, St. Louis, MO, USA). RNA samples were purified using a RNA Clean & Concentrator kit (Zymo research, R1017, Irvine, USA) to remove genomic DNA. The quantity and quality of RNA were evaluated using a DS-11 Series Spectrophotometer / Fluorometer (DeNovix, Wilmington, USA). The cDNA was synthesized using the SYBR PrimeScript reverse transcription PCR (RT-PCR) kit II (Takara, RR037A, Otsu, Shiga, Japan). To decipher the expression profile of Atg genes in different developmental stages of I. scapularis, qPCR was employed to detect expression levels in eggs, larvae, nymphs, and adults (female and male). We used the Mx3005P Real-Time system (Stratagene, La Jolla, USA) with SYBR green detection (Agilent Technologies, 600830, Santa Clara, USA). All protocols were according to the manufacturer’s instructions. For qPCR, we used the glyceraldehyde 3-phosphate dehydrogenase and tubulin endogenous genes as controls. Primers used in this study are listed in Supplementary Table S3.
Wang X.R., Kurtti T.J., Oliver J.D, & Munderloh U.G. (2020). The identification of tick autophagy related genes in Ixodes scapularis responding to amino acid starvation. Ticks and tick-borne diseases, 11(3), 101402.
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3

Quantifying Gene Expression via Northern Blot and qPCR

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Total cellular RNA was isolated using TRIzol (Invitrogen) or TRIReagent (Genbiotech) using the manufacturer’s protocols. mRNAs were reverse-transcribed using oligo-dT primers (Biodynamics). Real-time qPCR was performed using a Stratagene MX 3000P QPCR with SYBR Green detection (Agilent). For real-time qPCR, 2 μg of total RNA was subjected to reverse transcription. A dilution of all samples was then used to measure the expression of the endogenous fos, using a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) product as normalizer. For Northern blots, 30 μg of RNA extracted were ran in a 1% agarose gel in TAE (Tris base, acetic acid and EDTA buffer) 1×, transferred on to a positively charged nylon membrane (HyBond, GE Healthcare) and incubated with a fos-specific probe 32P-labelled by PCR.
Degese M.S., Tanos T., Naipauer J., Gingerich T., Chiappe D., Echeverria P., LaMarre J., Gutkind J.S, & Coso O.A. (2015). An interplay between the p38 MAPK pathway and AUBPs regulates c-fos mRNA stability during mitogenic stimulation. The Biochemical journal, 467(1), 77-90.
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4

Quantifying Apoptosis Gene Expression

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Total RNA was isolated from cells using TRI reagent (Sigma) and purified using an RNA Clean & Concentrator kit (Zymo Research). The quantity and quality of RNA were evaluated using a DS-11 series spectrophotometer/fluorometer (DeNovix). cDNA was synthesized using the SYBR PrimeScript reverse transcription-PCR (RT-PCR) kit II (TaKaRa). The expression levels of apoptosis-related genes in different treatments were determined using qPCR on the Mx3005P real-time system (Stratagene) with SYBR green detection (Agilent Technologies). Primers used in this study are listed in Table S2.
Wang X.R., Burkhardt N.Y., Kurtti T.J., Oliver J.D., Price L.D., Cull B., Thorpe C.J., Thiel M.S, & Munderloh U.G. (2021). Mitochondrion-Dependent Apoptosis Is Essential for Rickettsia parkeri Infection and Replication in Vector Cells. mSystems, 6(2), e01209-20.
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