Quantitative RT-PCR (RT-qPCR) was performed with a standard procedure. Briefly, total RNA, with efficient recovery of small RNAs, was obtained using a miRCURY RNA isolation kit (Exiqon, Denmark), and cDNA was synthesised by a Universal cDNA Synthesis Kit (Exiqon). Detection of the mature form of the miRNAs was performed using the miRCURY LNA Universal RT microRNA PCR Kit and LNA microRNA Primer Sets, according to the manufacturer’s instructions (Exiqon). Relative expression levels were determined using the comparative quantification characteristic of the Rotorgene software. The U6 small nuclear RNA was used as an internal control and the comparative Ct (ΔΔCt) method was used to determine the expression fold change. A melting curve analysis was performed for each of the primer sets utilised, and each showed a single peak indicating the specificity of each of the primers tested.
Microrna lna primer sets
Manufactured by Qiagen
Sourced in Germany
The MicroRNA LNA™ primer sets are designed for the detection and quantification of microRNA (miRNA) expression. These primer sets utilize Locked Nucleic Acid (LNA) technology to provide enhanced specificity and sensitivity in miRNA analysis.
Automatically generated - may contain errors
Lab products found in correlation
Universal cdna synthesis kit, by Qiagen (4 mentions)
Mircury lna universal rt microrna pcr kit, by Qiagen (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Mircury rna isolation kit, by Qiagen (1 mentions)
Universal cdna synthesis kit 2, by Qiagen (1 mentions)
Step one plus real time pcr thermal cycler, by Thermo Fisher Scientific (1 mentions)
Sybr green master mix sybr premix ex taqii, by Takara Bio (1 mentions)
5 protocols using microrna lna primer sets
1
Quantitative RT-PCR for miRNA Expression
2
Quantifying miR-200 Family Expression
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Extraction of the total RNA, with effective recovery
of small RNAs, was done in the three cell lines using
miRCURY RNA isolation kit (Exiqon, Denmark). Then,
cDNA was synthesized using the Universal cDNA
Synthesis Kit (Exiqon, Denmark).
With regard to the company’s guideline, the mature form of miR-200family was detected using LNA microRNA Primer Sets and miRCURY LNA Universal RT microRNA
PCR Kit (Exiqon, Denmark). In the next step, relative levels of expression were identified
using the relative quantification feature of Rotorgene software. Then, U6 small nuclear
RNA was employed as an internal control. Afterwards, comparative Ct (ΔΔCt) technique was
applied for determining fold changes of expression. Finally, a melting curve was analyzed
for all of the utilized primer collections, all of which exhibited a single peak. They
represented specificity of the all experienced primers. All assessments were performed
three times.
of small RNAs, was done in the three cell lines using
miRCURY RNA isolation kit (Exiqon, Denmark). Then,
cDNA was synthesized using the Universal cDNA
Synthesis Kit (Exiqon, Denmark).
With regard to the company’s guideline, the mature form of miR-200family was detected using LNA microRNA Primer Sets and miRCURY LNA Universal RT microRNA
PCR Kit (Exiqon, Denmark). In the next step, relative levels of expression were identified
using the relative quantification feature of Rotorgene software. Then, U6 small nuclear
RNA was employed as an internal control. Afterwards, comparative Ct (ΔΔCt) technique was
applied for determining fold changes of expression. Finally, a melting curve was analyzed
for all of the utilized primer collections, all of which exhibited a single peak. They
represented specificity of the all experienced primers. All assessments were performed
three times.
3
Validating miRNA Expression Levels
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For validation of miRNA expression, Universal cDNA synthesis kit (Exiqon, Denmark) was used for cDNA synthesis. Housekeeping gene (SNORD48) and cDNA control were controlled with spike in primers. Of the results of cDNA’s Snord48 and spike, those that are between Ct 15–29 were worked in LightCycler 480 II (Roche, Germany) using microRNA LNA™ primer sets (Exiqon, Denmark) and ExiLENT SYBR® Green Master Mix kit for the specified miRNAs. Δ/Δ Ct method was used to carry out relative quantification for the acquired results. With this method, miRNA Ct values were normalized via snord48 and U6 house keeping genes (this value is denoted as Δ Ct) after which the groups were compared among themselves and the acquired result yielded Δ/Δ Ct. If this value is greater than 2, regulation result was determined as a Target positive expression increase and if it is less than -2, target down was determined as the result of it was controlled by the microarray data.
4
miRNA Expression Profiling via RT-qPCR
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Reverse transcription (RT) reaction was performed on 500 ng of the total RNA using the universal cDNA synthesis kit II (Exiqon, Vedbaek, Denmark) in a 10 μL reaction. The thermal cycling parameters were 60 minutes at 42 °C, 5 minutes at 95 °C for the heat-inactivation of the reverse transcriptase, and immediate cooling at 4 °C.
Subsequently, the real-time quantitative PCR (RT-qPCR) of hsa-miR-17-5p, hsa-miR-361-5p, hsa-miR-885-5p, and the candidate normalizer hsa-miR-191-5p was performed in triplicate with the specific primers on the Step One Plus Real-Time PCR thermal cycler (Applied Biosystems, Foster City, CA, USA) using SYBR Green Master Mix: SYBR Premix Ex TaqII (TaKaRa, Tokyo, Japan) and microRNA LNA™ primer sets (Exiqon, Vedbaek, Denmark); in accordance with the manufacturer’s instructions. The thermal cycling parameters were 10 minutes at 95 °C; this was followed by 40 cycles of 10 seconds at 95 °C and 1 minute at 60 °C.
Relative expression levels were calculated using the comparative CT method with hsa-miR-191-5p as the normalizing control.
Subsequently, the real-time quantitative PCR (RT-qPCR) of hsa-miR-17-5p, hsa-miR-361-5p, hsa-miR-885-5p, and the candidate normalizer hsa-miR-191-5p was performed in triplicate with the specific primers on the Step One Plus Real-Time PCR thermal cycler (Applied Biosystems, Foster City, CA, USA) using SYBR Green Master Mix: SYBR Premix Ex TaqII (TaKaRa, Tokyo, Japan) and microRNA LNA™ primer sets (Exiqon, Vedbaek, Denmark); in accordance with the manufacturer’s instructions. The thermal cycling parameters were 10 minutes at 95 °C; this was followed by 40 cycles of 10 seconds at 95 °C and 1 minute at 60 °C.
Relative expression levels were calculated using the comparative CT method with hsa-miR-191-5p as the normalizing control.
5
Validating miRNA expression with qRT-PCR
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For validation of miRNA expression, Universal cDNA Synthesis kit (Exiqon, Denmark) was used for cDNA synthesis. Housekeeping gene (SNORD48) and cDNA control were controlled with a spike in primers. Of the results of cDNA's Snord48 and Spike, those that are between Ct values 15 and 29 regarding Ct values were worked in LightCycler 480 II (Roche, Germany) using microRNA LNA™ primer sets (Exiqon, Denmark) and ExiLENT SYBR® Green Master Mix kit for the specified miRNAs. Δ/ΔCt method was used to carry out relative quantification for the acquired results. With this method, miRNA Ct values were normalized via Snord48 and U6 housekeeping genes (this value is denoted as ΔCt) after which the groups were compared with themselves and the acquired result yielded Δ/ΔCt and if this value is greater than 2, regulation result was determined as a target positive expression increased and if it is less than −2, target down was determined as the result of being controlled by the microarray data.
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