Sorted cells were lysed in the TRIzol reagent (Ambion, Grand Island, NY, USA). Total RNA was then purified and further cleaned up using the RNeasy Micro Kit (Qiagen, Hilden, Germany). Reverse transcription was performed using the SuperScriptTM II kit (Invitrogen, Frederick, MA, USA) following the manufacturer’s instructions. The cDNA samples were then subjected to real-time PCR using Sybr-green reagents and an Eppendorf RealPlex 4 cycler. The gapdh expression was used as an internal control. The reaction specificity was determined by product melting curves. The PCR products were verified by running 3% agarose gels. Data were analyzed by ΔΔ threshold cycle method and presented as Fold Changes. The primer sequences were listed in Table 1 .
Realplex4 cycler
Manufactured by Eppendorf
Sourced in Germany, United States
The Realplex4 cycler is a real-time PCR instrument designed for quantitative analysis of nucleic acid samples. It features four independent sample blocks, allowing for simultaneous processing of multiple experiments. The Realplex4 cycler provides accurate temperature control and reliable data acquisition for real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green pcr master mix, by Thermo Fisher Scientific (4 mentions)
Adhesivecap tube, by Zeiss (2 mentions)
P a l m laser microdissection system, by Zeiss (2 mentions)
Rneasy ffpe kit, by Qiagen (2 mentions)
Membrane pen slides, by Zeiss (2 mentions)
Thermoscript reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Superscripttm 2 kit, by Thermo Fisher Scientific (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Rneasy micro kit, by Qiagen (1 mentions)
Faststart taqman probe master mix, by Roche (1 mentions)
5 protocols using realplex4 cycler
1
RNA Extraction and qRT-PCR Analysis
2
EGFP-labeled Neuron Transcriptome Analysis
3
Quantitative Analysis of miRNA and mRNA
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miR and mRNA RT-qPCR was performed with Fast Start Universal SYBR Green Master Mix (Roche-Genentech, San Francisco, CA, USA) on an Eppendorf Realplex 4 cycler (Hamburg, Germany). Forward primers for specific mature miRs were designed as described in the microScript miR cDNA synthesis handbook and using miRBase 21 as a sequence reference (Table S3 ). The following program was used for miR RT-qPCR: (1) 95 °C for 10 min, (2) 95 °C for 15 s, (3) 60 °C for 30 s, (4) 72 °C for 15 s, and melting curve analysis (Table S3 ). The following program was used for mRNA RT-qPCR: (1) 95 °C for 10 min, (2) 95 °C for 15 s, (3) 60 °C for 60 s, and melting curve analysis. Ribosomal protein lateral stalk subunit P1 (Rplp1) and U87 were used to normalize the data for analysis for mRNA and miR RT-qPCR, respectively. miR and mRNA expression was analyzed using the ΔΔCt method.
4
EGFP-labeled Neuron Transcriptome Analysis
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35 μm sections of labeled brains were stained for EGFP, and mounted on membrane PEN slides (Carl Zeiss). Labeled neurons were visualized, and captured into adhesive cap tubes (Carl Zeiss) using a PALM laser microdissection system (Carl Zeiss) using a uv laser to cut and flip tissue samples directly into inverted adhesive caps placed above slides. RNA was extracted using an RNeasy FFPE kit (QIAGEN). Reverse transcription was performed using random hexamers and Thermoscript reverse transcriptase (Fisher Scientific). Real time PCR was performed using SYBR® Green PCR Master Mix (Thermo Fisher) on a Realplex4 cycler (Eppendorf). We confirmed the specificity and size of the amplicons by running the PCR product on agarose gels, and by melting curve analysis. For Klhl14-overexpression experiments, data were analyzed using an unpaired two-sided t test. The PCR primers are listed in Key Resources Table .
5
Quantification of Gardnerella vaginalis
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Gardnerella vaginalis-specific qPCR targeting the cpn60 gene was performed using an Eppendorf Realplex4 cycler to quantify organism load, compared with a plasmid standard curve (pGEMT-easy(cpn60)), as previously described with modifications:91 (link) each reaction contained 0.25 nmol of probe, 0.5 nmol of gene-specific primers, 1 μl of extracted DNA, and 5 μl of 2XFastStart TaqMan Probe Master mix (Roche, USA) in a total of 10 μl. Results were expressed as copies of microorganism per sample.
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