For qRT-PCR, total RNA was individually extracted from each EPS sample. The first-strand miRNA-cDNA PCR template was generated from total RNA using the TaqMan® MicroRNA Reverse Transcription Kit (Applied Biosystems), including an artificial RNA spike-in (cel-mir-39) as loading control [27 (link), 33 (link)]. The cDNA was then used in PCR on a 7500HT real-time PCR instrument (Applied Biosystems). Triplicate samples, and inter-assay controls were used throughout. For each assay, the Ct (Cycle threshold) of miRNA was subtracted from the average cel-mir-39 Ct value to obtain a ΔCt value. The relative folds were calculated utilizing the 2(-ΔCt) method.
7500 ht real time pcr instrument
Manufactured by Thermo Fisher Scientific
The 7500 HT real-time PCR instrument is a laboratory equipment used for amplifying and detecting specific DNA sequences in a sample. It can perform real-time polymerase chain reaction (PCR) analysis to quantify target nucleic acid sequences. The instrument provides accurate and reliable results for a variety of applications.
Automatically generated - may contain errors
Lab products found in correlation
Taqman microrna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Taqman gene expression master mix, by Thermo Fisher Scientific (2 mentions)
Omniscript rt kit, by Qiagen (2 mentions)
Taqman universal master mix 2, by Thermo Fisher Scientific (2 mentions)
Gapdh, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
C myc, by Thermo Fisher Scientific (1 mentions)
Hnrnpu, by Thermo Fisher Scientific (1 mentions)
Taqman mir assay, by Thermo Fisher Scientific (1 mentions)
Smpd3, by Thermo Fisher Scientific (1 mentions)
5 protocols using 7500 ht real time pcr instrument
1
Quantifying miRNA Levels by qRT-PCR
2
Quantitative Real-Time PCR for mRNA and pri-miRNA Expression
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For mRNA and pri-miR expression analysis, 0.5 μg RNA was reversely transcribed to cDNA by use of the Omniscript RT Kit (Qiagen, Cat. # 205111) with Primer “random” (Roche, Cat. # 11034731001), following the manufacturer’s protocol. The cDNA was amplified using 1 µL of the cDNA preparation, the Taqman Gene Expression Master Mix (Applied Biosystems, Cat. # 4440040), and Taqman probes for hnRNPU, c-myc, GAPDH, and 18S (all from Thermo Fisher Scientific: hnRNPU Cat. # 4331182, Assay ID Hs00244919_m1; GAPDH, Cat. # 4331182, Assay ID Hs02786624_g1; c-myc, Cat. # 4453320, Assay ID Hs00153408_m1; 18S, Cat. # 4331182, Assay ID Hs99999901_s1; pri-miR-30c-1, Cat. # 4427012, Assay ID Hs03303371_pri; pri-miR-30c-2 Cat. # 4427012, Assay ID Hs03302833_pri) in a 7500 HT real-time PCR instrument (Applied Biosystems). GAPDH was used as an internal control and relative expression was calculated as ΔΔCT values.
3
Quantification of Extracellular Vesicle miRNA
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10 ng of total RNA were used for cDNA preparation with the TaqMan microRNA Reverse Transcription kit (Applied Biosystems, Cat# 4366596), adhering to the manufacturer’s protocols. 1 µL of the resulting cDNA solution was used for quantitative real-time PCR with the respective TaqMan probe (hsa-miR-126-3p Cat# 4427975, Assay ID 002228; hsa-miR-222-3p, Cat# 4427975, Assay ID 000525; hsa-miR-223-3p, Cat# 4427975, Assay ID 000526; cel-miR-39-3p, Cat# 4427975, Assay ID 000200; RNU6b, Cat# 4427975, Assay ID 001093) and TaqMan Universal Master Mix II (Applied Biosystems, Cat# 4440040) in a 7500 HT Real-Time PCR instrument (Applied Biosystems). MiR levels in pEVs were calculated as 2−ddCT vs. the spike-in control cel-miR-39. For the experiments with HCAECs, cellular miR-expression was calculated as 2−ddCT vs RNU6b as an internal control.
4
Quantification of Exosomal microRNAs
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MiRs were quantified with Taqman miR-Assays (all from Thermo Fisher Scientific: hsa-miR-30c Cat. # 4427975, Assay ID 000419; hsa-let-7d, Cat. # 4427975, Assay ID 002283; hsa-miR-20a, Cat. # 4427975, Assay ID 000580; hsa-miR-125a-3p, Cat. # 4427975, Assay ID 002199; RNU44, Cat. # 4427975, Assay ID 001094; snRNA U6 Cat. # 4440887, Assay ID 001973) in a 7500 HT Real-Time PCR instrument (Applied Biosystems). 10 ng of total RNA was reversely transcribed to cDNA by use of the TaqMan microRNA Reverse Transcription kit (Applied Biosystems, Cat. # 4366597), according to the manufacturer’s protocols. Then, quantitative real-time PCR was performed in triplicate using the TaqMan Universal Master Mix II (Applied Biosystems, Cat. # 4440040) and 1 µL of the cDNA solution after reverse transcription, which represents the equivalent of 0.67 ng RNA. Relative expression of miR-30c-5p, let-7d-5p and miR-20a-5p was calculated as ΔΔCT values, with RNU-44 as an internal control. Absolute miR expression was calculated by use of a concentration gradient of miR-30c-5p-mimic (Thermo Fisher Scientific, Cat. # 4464066 Assay ID MC11060) ranging from 6.67 × 10−10 to 6.67 × 10−17 g RNA and corresponding CT values from 6 to 39. MiR levels in EV-recipient cells were quantified after 24 h of incubation with EVshnRNPU kd/EVsControl.
5
RNA Isolation and qRT-PCR Analysis of Sphingolipid Genes
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RNA was isolated with Trizol (Invitrogen, Cat# 15596026) and chloroform, as previously described [47 ]. In brief, the cells were lysed in Trizol, after washing with ice-cold PBS. RNA was isolated with chloroform, precipitated with isopropanol, washed twice with ethanol, dried, and resuspended in water. The quality and concentration of the RNA were assessed using a Nanodrop2000 (Thermo Fisher Scientific).
For reverse transcription, we used 0.5 μg RNA and the Omniscript RT Kit (Qiagen, Cat# 205111) with a “random” primer (Roche, Cat# 11034731001), following the manufacturer’s protocol. For real-time PCR, we diluted 20 µl cDNA preparation with 480 µl water and used 9 µl of the dilution together with 11 µl of the Taqman Gene Expression Master Mix (Applied Biosystems, Cat# 4440040), and the respective Taqman probes for SMPD1, SMPD2, SMPD3, CERS5, CERS6, and GAPDH (all from Thermo Fisher Scientific: SMPD1 Cat# 4331182, Assay ID Hs04183448_m1; SMPD2, Cat# 4331182, Assay ID Hs00162006_m1; SMPD3, Cat# 4331182, Assay ID Hs00920354_m1; CerS5, Cat# 4331182, Assay ID Hs00332291_m1; CerS6, Cat# 4331182, Assay ID Hs00826756_m1; GAPDH, Cat# 4331182, Assay ID Hs02786624_g1) in a 7500 HT real-time PCR instrument (Applied Biosystems). We used GAPDH as an internal control and relative expression was calculated as 2−ΔΔCT.
For reverse transcription, we used 0.5 μg RNA and the Omniscript RT Kit (Qiagen, Cat# 205111) with a “random” primer (Roche, Cat# 11034731001), following the manufacturer’s protocol. For real-time PCR, we diluted 20 µl cDNA preparation with 480 µl water and used 9 µl of the dilution together with 11 µl of the Taqman Gene Expression Master Mix (Applied Biosystems, Cat# 4440040), and the respective Taqman probes for SMPD1, SMPD2, SMPD3, CERS5, CERS6, and GAPDH (all from Thermo Fisher Scientific: SMPD1 Cat# 4331182, Assay ID Hs04183448_m1; SMPD2, Cat# 4331182, Assay ID Hs00162006_m1; SMPD3, Cat# 4331182, Assay ID Hs00920354_m1; CerS5, Cat# 4331182, Assay ID Hs00332291_m1; CerS6, Cat# 4331182, Assay ID Hs00826756_m1; GAPDH, Cat# 4331182, Assay ID Hs02786624_g1) in a 7500 HT real-time PCR instrument (Applied Biosystems). We used GAPDH as an internal control and relative expression was calculated as 2−ΔΔCT.
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