Three different batches of embryos were electroporated with KDM4B-MO unilaterally at stage 8 and incubated to stage 13. Otic placodes from the injected and the uninjected side were dissected separately. Total RNA was extracted using an RNAqueous-Micro isolation kit (Ambion) according to the manufacturer’s instruction. After DNaseI-amplification grade (Invitrogen) treatment, 1 µg of total RNA was used for cDNA synthesis using SuperScriptII (Invitrogen) and random hexamers (Roche). qPCR was performed using a 96-well plate in MX3005P equipment (Agilent Technologies). Each reaction was performed using Fast Start Syber green (Roche) in a 15-µl volume. All the used primers (Table S1) were tested to have amplification efficiency ranging from 95 to 100%. The subsequent quantification method was done using the ΔΔCt method (threshold cycle). For a reference gene, we used a qPCR primer designed for the GAPDH gene. Gene expression levels are represented relative to GAPDH expression.
Rnaqueous micro isolation kit
Manufactured by Thermo Fisher Scientific
The RNAqueous-Micro isolation kit is a product designed for the extraction and purification of total RNA from small sample sizes. It provides a reliable and efficient method for isolating high-quality RNA from limited biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Dnase 1 amplification grade, by Thermo Fisher Scientific (2 mentions)
Random hexamer, by Roche (1 mentions)
Faststart sybergreen, by Roche (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Quantstudio 6 flex real time pcr system, by Thermo Fisher Scientific (1 mentions)
Retroscript, by Thermo Fisher Scientific (1 mentions)
Facsdiva v8, by BD (1 mentions)
Facsaria cell sorter, by BD (1 mentions)
Superscript 2 kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
6 protocols using rnaqueous micro isolation kit
1
Quantifying KDM4B Knockdown Effects
2
Quantitative Gene Expression Analysis
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Total RNA was extracted from individual larvae using the RNAqueous-Micro Isolation Kit (Ambion). cDNA was made using oligo dT primer and SuperScript IV reverse transcriptase (Invitrogen). qPCR was performed on the Applied Biosystems QuantStudio Flex 6 Real-Time PCR System using probe-based assays or SYBR Green. The delta–delta ct method was used to determine the relative levels of mRNA expression between experimental samples and controls. ef1a was used as the reference gene for determination of relative expression of all target genes. Sequences of the qPCR probes and primer sets used in qPCR and RT-PCR analyses are listed in Table 1 .
3
Quantifying Human Placental Cell mRNA
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Using an RNAqueous-Micro isolation kit (Ambion, Austin, TX), total RNA was isolated from primary cultures of human TDCs, CTBs, and STBs as well as choriodecidual explant cultures, per manufacturer's instructions. Reverse transcription was performed using the Retroscript (Invitrogen), as follows: template RNA and random primers were incubated at 65 C for 5 minutes (to eliminate any secondary structures). After adding the buffer and enzyme, the reaction was performed at 42 C for 1 hour. qPCR was performed using TaqMan gene expression assays for CSF2, CSF2RA, matrix metalloproteinase (MMP) 3, 7, or 9, as well as IL-1B, IL-6, or IL-8 (Applied Biosystems, Foster City, CA) (Table 2). All samples were run in triplicate. Expression of the target mRNAs was normalized to b-actin mRNA levels and the 2 ÀDDCT (cycle threshold) method was used to calculate relative expression levels.
4
Zebrafish Cell Sorting and RNA Extraction
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Dissociation of pools of 25–50 2.5 dpf transgenic zebrafish embryos either carrying the macrophage reporter (mpeg1:EGFP) or the erythrocyte reporter (gata1:DsRed) was conducted using a protocol adapted from [75 (link)]. Fluorescently tagged cells were sorted from non-fluorescent cells using the BD FACSAria cell sorter equipped with three lasers. Non-fluorescent control embryos were used for setting gates in FACS for GFP and DsRed positive cells. BD FACSDiva V8.0.1 software was used for cell sorting and FACS plots. Total RNA was extracted from the different populations of cells (GFP positive, GFP negative, DsRed positive, DsRed negative) using the RNAqueous-Micro Isolation Kit (Ambion), and made into cDNA for RT-PCR analyses as described above.
5
Trigeminal Ganglion sEV miRNA Profiling
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RNA was prepared from the trigeminal ganglion and sEV fraction using the RNAqueous-Micro isolation kit (Ambion) following the manufacturer’s instructions, and RNA was treated with amplification-grade DNaseI (Invitrogen). The reverse transcription reaction to obtain the cDNA was performed with the MystiCq® microRNA cDNA Synthesis Mix kit (Merck) and amplified by PCR using the following primers (miR-34–5p Fw: 5’-GCC GCT GGC AGT GTC TTA G-3’; miR-203 Fw: 5’-CCG GCG TGA AAT GTT TAG G-3’; and miR-UNI Rev: 5’-GAG GTA TTC GCA CCA GAG GA-3’).
6
Quantifying Gene Expression in DCs and Trophoblasts
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Total RNA from term DCs and trophoblasts was isolated and purified using a RNAqueous-Micro isolation kit, according to the manufacturer's instructions (Ambion, Austin, TX). Reverse transcription was performed using the Su-perScript II kit (Invitrogen, Carlsbad, CA) in two steps: template RNA and random primers were incubated at 65 C for 5 minutes to eliminate any secondary structures, and then the buffer and enzyme were added and the reaction was performed at 42 C for 1 hour. Reverse transcriptase was inactivated at 70 C for 10 minutes. Real-time quantitative PCR (qPCR) was performed with TaqMan gene expression assays for FKBP51, FKBP52, vimentin, IL-1b, and the reference gene, b-actin (Applied Biosystems, Foster City, CA). Standard curves were generated from serial dilutions of a known sample for each assay. All samples were run in triplicate, and the average was used for each sample. The 2 ÀDDC T (cycle threshold) method was used to calculate relative expression levels. Results were reported as a fold change (Table 1).
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