For siRNA transfection studies, cells were allowed to grow at 60–80% confluency and transfected with 100 μM each of scrambled siRNA (EHUEGFP, esiRNAs, Sigma Aldrich) and siRNAs specifically targeting KDM5A (EHU0112051, esiRNAs, Sigma Aldrich) and LSD2 (EHU052581, esiRNAs, Sigma Aldrich) using 5 μl of lipofectamine 2000 (Invitrogen). After 6 hours, the culture medium was replaced with fresh medium to reduce the lipofectamine-mediated toxicity. Transfections were performed as per the manufacturer’s instructions in 6-well and 96-well plate. The cells were then cultured for 48 hours. After that, total RNA was isolated using TRIzol reagent and LSD2 and KDM5A mRNA levels were determined using real-time PCR.
Esirna
Manufactured by Merck Group
Sourced in United States
EsiRNA is a laboratory instrument designed for the efficient synthesis and purification of small interfering RNA (siRNA) molecules. It utilizes an automated process to streamline the production of siRNA samples for research applications.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions)
Esirna, by Thermo Fisher Scientific (8 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (8 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (5 mentions)
On targetplus smartpool, by Horizon Discovery (4 mentions)
Ehuegfp, by Merck Group (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Rnaimax transfection reagent, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (2 mentions)
Profection mammalian transfection system, by Promega (2 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (2 mentions)
Dharmafect, by Horizon Discovery (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Silencer select sirna, by Thermo Fisher Scientific (1 mentions)
Pcmv6 an 3ddk, by OriGene (1 mentions)
Hyperfect reagent, by Qiagen (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Oligofectamine rnai max, by Thermo Fisher Scientific (1 mentions)
Powerfect, by Tebu-Bio (1 mentions)
X tremegene hp dna transfection reagent, by Merck Group (1 mentions)
40 protocols using esirna
1
Silencing KDM5A and LSD2 via siRNA
2
Genetic Manipulation of Human DNA Repair Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The mammalian expression vector containing the hSSB1 CDS (pCMV6-AN-3DDK) was supplied by Origene. Site-directed mutagenesis (SDM) was used to introduce the non-coding mutations for small interfering RNA (siRNA) resistance and was performed using the polymerase Pfu Ultra (Stratagene). The pET28a hSSB1 vector has been described previously. For the preparation of truncation mutants, premature STOP codons were introduced by SDM as per above. The preparation of hOGG1 point mutants has been performed on pGEX-hOGG1 vector, as described earlier. Primer sequences are listed in Supplementary Table S1.
Mammalian expression vectors were transfected using Lipofectamine 2000 (Life Technologies).
Stealth siRNA against hSSB1 were synthesized by Life technologies (Invitrogen). Individual siRNA sequences were (sense) 5′-GACAAAGGACGGGCAUGAGdTdT and (antisense) 5′-CUCAUGCCCGUCCUUUGUCdTdT (17 (link)). hOGG1 was targeted using either pooled esiRNAs (Sigma Aldrich) or the Silencer Select siRNA sequences (sense) 5′-GAUCAAGUAUGGACACUGAtt and (antisense) 5′-UCAGUGUCCAUACUUGAUCcg (Life Technologies). siRNAs were transfected using RNAiMax (Life Technologies).
Mammalian expression vectors were transfected using Lipofectamine 2000 (Life Technologies).
Stealth siRNA against hSSB1 were synthesized by Life technologies (Invitrogen). Individual siRNA sequences were (sense) 5′-GACAAAGGACGGGCAUGAGdTdT and (antisense) 5′-CUCAUGCCCGUCCUUUGUCdTdT (17 (link)). hOGG1 was targeted using either pooled esiRNAs (Sigma Aldrich) or the Silencer Select siRNA sequences (sense) 5′-GAUCAAGUAUGGACACUGAtt and (antisense) 5′-UCAGUGUCCAUACUUGAUCcg (Life Technologies). siRNAs were transfected using RNAiMax (Life Technologies).
3
Transfection of Plasmids, LNAs, and siRNAs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Plasmids were transfected (or co-transfected) with Lipofectamine 2000 (Thermo Scientific). LNAs targeting miRNAs (Exiqon) and siRNAs targeting G4 driver genes (Qiagen) were transfected with Hyperfect reagent (Qiagen) at a concentration of 20 nM (each molecule). EsiRNAs (SIGMA Aldrich) were used as in [11 (link)] for linc-NeD125 knockdown.
4
esiRNA Knockdown of BATF3 in U2OS Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For esiRNA knockdown experiments of BATF3, 10.000 U2OS cells stably expressing GRα [44 (link)] or GRγ [22 (link)] were seeded per well of a 48-well plate. The next day, cells were transfected with 50nM esiRNAs (Sigma-Aldrich) against BATF3 (EHU153831) or a non-target esiRNA control (RL, EHURLUC) using lipofectamine 2000 (Invitrogen). 6 h after transfection, cells were washed once and re-fed with DMEM/5% FBS. 48 h past transfection, cells were treated for 4 h with 1 μM dexamethasone or ethanol as vehicle control to measure the effect of BATF3 knockdown on GR-dependent regulation of endogenous target genes. RNA was isolated using an RNeasy kit (Qiagen) with on-column DNAse digestion prior to reverse transcription and analysis by Quantitative Real Time PCR using the primers listed in Table 2 .
5
esiRNA Knockdown of SFPQ and NONO in Transgenic Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For esiRNA knockdown experiments, 10.000 transgenic cells were seeded per well of a 48-well plate. The next day, these cells were transfected with a mix of 75 ng of each esiRNAs (Sigma-Aldrich) against SFPQ and NONO (EHU 158661, EHU 071361) or a non-target esiRNA control (RL) using lipofectamine 2000 (Invitrogen). Thirty two hours post transfection, cells were treated for 16 h with 1 μM dexamethasone, or 0.1% ethanol as vehicle control. Cells were lysed and luciferase activity was measured.
6
Transient Gene Silencing Experiments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transient gene-silencing experiments were performed with endoribonuclease-prepared siRNAs (esiRNA, Sigma) for CPS1 and CAD, and with ON-TARGETplus-SMART pools (Dharmacon) for LKB1, CREB1, FOXA1, TEAD4, ODC, TSC-1 and -2, AMPKα-1 and -2. Briefly, siRNA oligos were transfected into cells with RNAi max transfection reagent (Invitrogen); esiRNA oligos targeting EGFP or siRNA Universal Negative Controls were used as a negative control (Sigma). For Extended Data Fig. 6e and 10d , triple transfection was performed (every other day, repeated three times) and western blots were assayed 144 hr after the first transfection. Viability assays were performed after 96 hr and cell death analyses and all other western blots were performed after 48 hr. BrdU incorporation was measured after 24 hr in H460 cells and after 36 hr in H2122 cells. For inducible RNAi experiments, shREN and shCPS1#1 and #2 were induced using doxycycline concentrations of 1.0–2.0 mg/ml.
7
Secondary Screen for Cytotoxic Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
55 genes identified in the original screen were tested in a secondary screen using endoribonuclease-prepared siRNAs (esiRNA) (Sigma-Aldrich). esiRNA transfection conditions were optimized with an esiRNA targeted to firefly luciferase (FLUC) in UMUC3 cells stably expressing FLUC. Knockdown efficiency was confirmed by qRT-PCR for 5 genes and esiRNA knockdown yielded >75% knockdown for all genes. Each transfection was done in triplicate in a 96-well plate with 500 cells, 24 ng/well esiRNA and 0.2 μL oligofectamine RNAi max (Life Technologies)/well. Media was replaced after 24 hours; cytotoxicity assays were done 48 hours post-transfection. Cytotoxicity was normalized to firefly luciferase (FLUC) transfected controls.
8
FOXO Knockdown in Breast Cancer Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MDA‐MB‐468 cells were grown to log phase in DMEM with 10% FBS without antibiotics. BT549 cells were grown to log phase in DMEM with 10% FBS without antibiotics. Cells were transfected with esiRNA (Sigma, St. Louis, MO, USA) FOXO1 (EHU156591), FOXO3 (EHU113611 ), FOXO4 (EHU075731 ), or EGFP control esiRNA (EHUEGFP) using Lipofectamine 3000 (utilized only L3000 reagent, Invitrogen, Carlsbad, CA, USA). FOXO1 RNAi or control was from Cell Signaling Technologies (cat: 6256 and 6568, respectively; Danvers, MA, USA) for BT549 cells in Fig. 4B ; Sigma FOXO1 esiRNA‐treated samples had the same gene expression results in BT549 cells (Fig. S3 ).
9
Lentiviral Transfection for NK Cell Manipulation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
293T cells were seeded and incubated at 37°C in a 5% CO2 environment until the cells were 60–80% confluent. The cells were transfected with plasmids using Lipofectamine 3000 Reagent (Fisher Scientific). NK cells were transfected with esiRNA (Sigma) using the nucleofection method (Lonza)41 (link). To generate lentivirus to infect human NK cells, 293T cells were co-transfected with pCDH expressing XBP1u, XBP1s, myrAKTΔ4−129, or pLKO.1 expressing XBP1 or AKT1 shRNA or corresponding control plasmids with the packaging constructs pCMV-VSVG and pCMV-ΔR9 by a ProFection® Mammalian Transfection System (Promega). The protocols for virus production and infection were modified from our previous reports42 (link),43 (link). During the lentiviral infection process, IL-15 was introduced into the cell culture to maintain survival of NK cells.
10
Reverse Transfection of mES and EpiLC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
At day 0: mES cells (105 cells/cm2) in 2i + LIF were reverse‐transfected with a mix of 200 ng esiRNA (100 ng/μL, Sigma) and 2 μL PowerFect (Tebu‐Bio, Boechout, Antwerp, Belgium,PowerFect_TM_SIRNA_TRANSFECTION_REAGENT.html">https://www.tebu-bio.com/Product/189SL100569-0.1ml/PowerFect_TM_SIRNA_TRANSFECTION_REAGENT.html ) in 110 μL buffer solution/well of a 0.1% gelatin‐coated 12‐well plate. After 6 hours at 37°C, the cells were replated in either N2B27 (for differentiation) or N2B27 + LIF + BMP4.
At day 2: EpiLCs (6 × 104 cells/cm2) were reverse‐transfected with 100 ng (100 ng/μL) esiRNA and 1 μL PowerFect in 55 μL buffer per well of a GFR Matrigel coated 24‐well plate. After 6 hours at 37°C, the cells were replated in N2B27 medium.
In both transfection conditions, the cells were lysed and RNA was harvested 48 hours post‐transfection, that is, as BMP4 + LIF, EpiLC, or NP cell state. In addition, for each sample the endogenous mRNA levels 6 hours post‐transfection were analyzed by RT‐qPCR. A knockdown is scored sufficient if at least a 50% reduction of the transcript was observed.
+ Expand
At day 0: mES cells (105 cells/cm2) in 2i + LIF were reverse‐transfected with a mix of 200 ng esiRNA (100 ng/μL, Sigma) and 2 μL PowerFect (Tebu‐Bio, Boechout, Antwerp, Belgium,
At day 2: EpiLCs (6 × 104 cells/cm2) were reverse‐transfected with 100 ng (100 ng/μL) esiRNA and 1 μL PowerFect in 55 μL buffer per well of a GFR Matrigel coated 24‐well plate. After 6 hours at 37°C, the cells were replated in N2B27 medium.
In both transfection conditions, the cells were lysed and RNA was harvested 48 hours post‐transfection, that is, as BMP4 + LIF, EpiLC, or NP cell state. In addition, for each sample the endogenous mRNA levels 6 hours post‐transfection were analyzed by RT‐qPCR. A knockdown is scored sufficient if at least a 50% reduction of the transcript was observed.
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
Revolutionizing how scientists
search and build protocols!