The largest database of trusted experimental protocols

Sirnas

Manufactured by Eurofins
Sourced in Luxembourg, Germany

SiRNAs are a class of small, double-stranded RNA molecules that play a role in the RNA interference (RNAi) pathway. They function by targeting and silencing specific mRNA molecules, thereby regulating gene expression.

Automatically generated - may contain errors

11 protocols using sirnas

1

siRNA Transfection in CHO-K1 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
siRNAs (Eurofins, Luxembourg) were transfected into CHO-K1 cells using the Neon® transfection system (Thermo Fisher Scientific) with the Neon® transfection system 100 µl kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. In brief, 5E6 cells were spun down (170 × g, 8 min) and resuspended in 100 µl buffer R. After the addition of 300 pmol of siRNAs, cells were shocked by applying one pulse with 1700 V and 20 ms. A mock transfection and a non-targeting siRNA (AllStars Negative Control siRNA, QIAGEN) were included as controls. Cells were allowed to recover in 10 ml media in Tubespin® bioreactors 50 (Techno Plastic products, Switzerland) for at least 2 hours post transfection without shaking at 37 °C, humidified air and 7% CO2. Afterwards, cultures were shaken at 250 rpm with a 12.5 mm shaking diameter.
+ Open protocol
+ Expand
2

siRNA Transfection by Electroporation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Small interference (si)RNAs were purchased from Eurofins MWG. Transfection was performed by electroporation as described [66 (link)]. Briefly, cells were trypsinized, washed once in RPMI medium, electroporated by using a Bio-Rad GenePulserX-Cell at 250 V and 800 μF and kept on ice for 30 min. Cells were then transferred to a 6 or 10 cm (Ø) culture dish and incubated for two days to allow the siRNA to downregulate the expression of its target. The effect of the siRNA was confirmed by Western blot and/or Q-RT-PCR analysis. The following siRNAs (sense-strand) were used: siBcl3 (5′-UGG UCU UCU CUC CGC AUC A-3′), siLuc (5′-CUU ACG CUG AGU ACU UCG A-3′), siIGFBP5 (5′-GCA GAU CUG UGA AUA UGA A-3′) and siSTAT3 (5′-GAA UCA CGC CUU CUA CAG A-3′).
+ Open protocol
+ Expand
3

Silencing PI4K Enzymes in HUVECs

Check if the same lab product or an alternative is used in the 5 most similar protocols
PI4KIIα and PI4KIIβ depletion was achieved by using siRNAs with the sequence 5′-GGAUCAUUGCUGUCUUCAA(TT)-3′ and 5′-GGUUCAAGUGGAAGUUACU(TT)-3′ (Eurofins/MWG), respectively. Mock transfections were performed in parallel without siRNA. HUVECs underwent two rounds of transfection with 300 pmol of siRNA per nucleofection (Amaxa Biosystems) as described previously (Lui-Roberts et al., 2008 (link)). Cells were assayed 2 days following the second round of nucleofection at 100% confluency.
+ Open protocol
+ Expand
4

Transient RNAi Silencing in Organism

Check if the same lab product or an alternative is used in the 5 most similar protocols
RNAi was performed by electroporating siRNAs as previously reported (Watanabe et al., 2014 (link)) with minor modifications. Briefly, 30 animals were electoporated as above either with a ‘scrambled’ siRNA (4 µM, diluted in water) used as control, or with a mixture of three siRNAs (Eurofins Genomics) targeted against p62/SQSTM1 or WIPI2 (final concentration 4 µM in water, see Table S5 for sequences). The procedure was repeated every other day as indicated.
+ Open protocol
+ Expand
5

Repeated siRNA Transfection of Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
Transfection of siRNAs was performed as described earlier (Klanert et al., 2019). Briefly, siRNAs (Eurofins, Luxembourg City, Luxembourg) were transfected using the Neon® transfection system (Thermo Fisher Scientific) with the Neon® transfection system 100 µl kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Therefore, 5 × 106 cells were centrifuged at 170 rcf for 8 min and then resuspended in 100 µl buffer R. After the addition of 300 pmol of siRNAs, cells were transfected by applying one pulse with 1,700 V and 20 ms. A mock transfection and a nontargeting “scrambled” siRNA (AllStars Negative Control siRNA; Qiagen, Venlo, The Netherlands) were included as controls. Cells were allowed to recover in 10 ml of the respective media in a 50‐ml TPP® Tubespin bioreactors for 1.5−2 hr post‐transfection without shaking at 37°C, humidified air and 7% CO2. Subsequently, cultures were shaken at 220 rpm. For the repeated transfections of cells with siRNAs, this procedure was repeated every 4th day. All siRNA sequences are provided in Table S2.
+ Open protocol
+ Expand
6

Transient Transfection of RAW264.7 Cells with siRNA Targeting cPLA2γ

Check if the same lab product or an alternative is used in the 5 most similar protocols
The procedure for transient transfection of RAW264.7 cells with siRNAs targeting group IVC phospholipase A2 (cytosolic phospholipase A2γ; cPLA2γ) was adapted from Valdearcos et al. [51 (link)]. Briefly, 3 × 105 cells were transfected with siRNAs (20 nM) (sequence: 5′-(GGAGGAGAGAGAGGAAGAGAA)TT-3′; Eurofins Genomics, Ebersberg, Germany) in the presence of 5 µl/mL LipofectamineTM RNAiMAX (Invitrogen) under serum-free conditions for 5 h. Afterward, 5% serum was added and the cells were maintained at normal culture conditions for 24 h. A scrambled siRNA was used as a negative control (sequence: 5′-UGGUUUACAUGUCGACUAA-3′).
+ Open protocol
+ Expand
7

Antibodies and siRNA for p53 Pathway

Check if the same lab product or an alternative is used in the 5 most similar protocols
The anti-p53 antibodies Pab421, Pab246, CM5 and 1C12 were purchased from Millipore (Darmstadt, Germany), Vector laboratories (Burlingame, CA, USA) and Cell signaling (Danvers, MA, USA), respectively. From Santa Cruz (Dallas, TX, USA), we obtained the antibodies against Oct-4 (C-10) Nanog (C-4), c-Jun (H79) and PCNA (PC10). Antibodies against acetylated and phosphorylated p53 (K379, S6, S15, S392) were from Cell signaling. Antibodies against GAPDH (6C5), PARC (PO69) and α-7 (MCP72) were from Hytest (Turku, Finland), BioLegend (San Diego, CA, USA) and Enzo (Loerrach, Germany), respectively. Antibodies against β-Actin and Histone H3 were purchased from Abcam (Cambridge, UK) and the antibody against MdmX (MDMX82) was from Sigma-Aldrich.
siRNAs were purchased from Eurofins (Ebersberg, Germany). Sequences are available on request. siRNA transfections were performed using Macsfectin (Miltenyi Biotec, Bergisch-Gladbach, Germany) following the manufacturer's instructions.
+ Open protocol
+ Expand
8

Silencing of TEAD, YAP/TAZ, GTSE1 and E2F1 in Breast Cancer Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols
siRNAs were purchased from Eurofins Genomics (sequences shown in Supplementary Table 1).
The AllStars negative control siRNA (Qiagen, 1027281) was used as negative control.
Lipofectamine RNAi-MAX (Invitrogen) was used for siRNAs transfections in antibiotic-free medium according to the manufacturer's instructions. Cells were transfected in reverse using 20nM siRNA. The TEAD1/3/4 and YAP/TAZ silencing was performed by transfecting the siRNAs in MDA-MB-231 and MDA-MB-157 cell lines for 72 hours.
GTSE1 and E2F1 silencing was carried out transfecting cells with siRNAs for 48 hours.
+ Open protocol
+ Expand
9

Oligonucleotide Synthesis and Labeling

Check if the same lab product or an alternative is used in the 5 most similar protocols
Synthetic single-stranded oligodeoxyribonucleotides (Supplementary Table S1) were purchased from Invitrogen, dissolved in bidistilled water and stored at −20°C until use. Concentrations were determined by spectrophotometry using extinction coefficients provided by the manufacturer. Radioactively 32P-5′-end-labeled oligonucleotides were desalted and separated from the remaining radioactively labeled ATP by gel-filtration chromatography (Bio-Spin 6 Columns, BIO-RAD). siRNAs (Supplementary Table S1) were purchased from Eurofins, dissolved in bidistilled water and stored at −20°C until use.
+ Open protocol
+ Expand
10

Cen-RNA Depletion by ASOs and siRNAs

Check if the same lab product or an alternative is used in the 5 most similar protocols
The Cen-RNA was knocked down by ASOs and siRNAs in different concentrations (Eurofins). The sequence for Cen-ASO was mU*mG*mU*mU*mU*T*T*C*A*G*T*G*T*A*A*mC*mU*mC*mA*mC. The sequence of specific 5MM-ASO was mU*mG*mA*mA*mU*T*A*C*A*G*T*G*A*A*A*mC*mA*mC*mA*mC (mismatched nucleotides were marked with bold and underline). The sequence for siRNA was GGA​AAC​GGG​AUU​UGU​AGA​ATT. The ASO to deplete MajSAT transcripts was designed against MajSAT RNAs: mC*mA*mG*mU*mU*T*T*C*T*T*G*C*C*A*T*mA*mU*mU*mC*mC. The control sequence also with five mismatches was mC*mA*mG*mA*mU*T*A*C*T*A*G*C*C*A*A*mA*mU*mA*mC*mC. The "m" represents 2′-O-methoxyethylribonucleotide. An asterisk represents a phosphorothioate bond.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!