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First siRNA library screening in hard-to-transfect HUVEC cells.

Zumbansen M, Altrogge LM, Spottke NU, Spicker S, Offizier SM, Domzalski SB, St Amand AL, Toell A, Leake D, Mueller-Hartmann HA - J RNAi Gene Silencing (2009)

Bottom Line: Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability.Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect.Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

View Article: PubMed Central - PubMed

Affiliation: Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany.

ABSTRACT
Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

No MeSH data available.


Primary Screen. HUVEC cells were transfected with 20 pmol of the combined Human siARRAY® SMARTpool® siRNA Libraries for Kinases (targeting 779 genes) and Cell Cycle Regulators (targeting 111 genes). Cell viability was analyzed 72 hrs post-Nucleofection®. (A) Representation of robust Z-scores of cell viability measures from 1 screening experiment. (B-E) Robust Z-scores of all primary hits with MAD of >3 in all three (B), two of three (C), or one of three (D) independent experiments and with MAD of <3 in all three experiments (E). (F) Robust Z-scores of the top 37 primary hits (with MAD of >3) from three independent experiments.
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Figure 2: Primary Screen. HUVEC cells were transfected with 20 pmol of the combined Human siARRAY® SMARTpool® siRNA Libraries for Kinases (targeting 779 genes) and Cell Cycle Regulators (targeting 111 genes). Cell viability was analyzed 72 hrs post-Nucleofection®. (A) Representation of robust Z-scores of cell viability measures from 1 screening experiment. (B-E) Robust Z-scores of all primary hits with MAD of >3 in all three (B), two of three (C), or one of three (D) independent experiments and with MAD of <3 in all three experiments (E). (F) Robust Z-scores of the top 37 primary hits (with MAD of >3) from three independent experiments.

Mentions: HUVEC Cells were transfected with siRNA pools targeting individual genes in the Human siGENOME® siRNA Libraries for protein kinases and cell cycle regulation. Multiple independent screening experiments (n=3) were performed to confirm the reproducibility of individual primary hits. Robust Z-score for cell viability was calculated for each of the 890 targets in the three independent experiments. As an example, the robust Z-scores of one screening experiment are shown in Figure 2A. A substantial proportion of targets displayed a median absolute deviation (MAD) below -3 or above 3 (MAD >3) including our positive controls PLK-1 and CHEK-1, which are members of both libraries. Thirty-five targets plus CHEK-1 and PLK-1 had a mean MAD greater than 3 in the three screening experiments and thus were considered as potential hits (Figure 2F). Eighteen of these targets showed a robust phenotype with MAD of greater than 3 in all three screening experiments (Figure 2B), while 24 targets were significant in two of the three experiments (Figure 2C) and 123 targets showed MAD greater than 3 in just one experiment (Figure 2D). The remaining 725 targets showed no response beyond the threshold in any experiment. The categories of targets with one or two experiments with MAD greater than 3 showed the highest standard deviations due to the occurrence of outliers. These categories also contained targets with a small standard deviation, suggesting that their importance for survival of HUVEC was not sufficiently strong for the chosen assay conditions (Figure 2B and C). Every sixth target fell into these categories of unclear importance, thus five of six targets were reproducibly classified as either important or not relevant for survival of HUVEC (Figure 2C and D). Generally, a considerable degree of variation was seen in the data, including the most significant hit category (Figure 2B). The most striking examples were MYC, which nevertheless could be validated, and CHEK1, which was a member of the library and also served as a positive control (Figure 2B and F). The degree of data variation might be attributed to not fully standardized cell culture conditions during the preparation of the experiments and thus argues for our strategy to select the primary hits from repeated screening experiments. As a consequence, we chose all 35 primary hits despite their standard deviation for further validation experiments. While a higher number of primary hits for validation lowered the probability for erroneously classified false negatives, it also lowered the validation rate. Of the 35 identified primary hits, 33 had a pro-proliferative/anti-apoptotic function, as their down-regulation led to increased cell death, while 2 had an anti-proliferate effect as their knockdown allowed for better cell viability. The 16 strongest of the 33 pro-proliferative hits plus the two positive controls were selected for further evaluation (Table 1).


First siRNA library screening in hard-to-transfect HUVEC cells.

Zumbansen M, Altrogge LM, Spottke NU, Spicker S, Offizier SM, Domzalski SB, St Amand AL, Toell A, Leake D, Mueller-Hartmann HA - J RNAi Gene Silencing (2009)

Primary Screen. HUVEC cells were transfected with 20 pmol of the combined Human siARRAY® SMARTpool® siRNA Libraries for Kinases (targeting 779 genes) and Cell Cycle Regulators (targeting 111 genes). Cell viability was analyzed 72 hrs post-Nucleofection®. (A) Representation of robust Z-scores of cell viability measures from 1 screening experiment. (B-E) Robust Z-scores of all primary hits with MAD of >3 in all three (B), two of three (C), or one of three (D) independent experiments and with MAD of <3 in all three experiments (E). (F) Robust Z-scores of the top 37 primary hits (with MAD of >3) from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2902142&req=5

Figure 2: Primary Screen. HUVEC cells were transfected with 20 pmol of the combined Human siARRAY® SMARTpool® siRNA Libraries for Kinases (targeting 779 genes) and Cell Cycle Regulators (targeting 111 genes). Cell viability was analyzed 72 hrs post-Nucleofection®. (A) Representation of robust Z-scores of cell viability measures from 1 screening experiment. (B-E) Robust Z-scores of all primary hits with MAD of >3 in all three (B), two of three (C), or one of three (D) independent experiments and with MAD of <3 in all three experiments (E). (F) Robust Z-scores of the top 37 primary hits (with MAD of >3) from three independent experiments.
Mentions: HUVEC Cells were transfected with siRNA pools targeting individual genes in the Human siGENOME® siRNA Libraries for protein kinases and cell cycle regulation. Multiple independent screening experiments (n=3) were performed to confirm the reproducibility of individual primary hits. Robust Z-score for cell viability was calculated for each of the 890 targets in the three independent experiments. As an example, the robust Z-scores of one screening experiment are shown in Figure 2A. A substantial proportion of targets displayed a median absolute deviation (MAD) below -3 or above 3 (MAD >3) including our positive controls PLK-1 and CHEK-1, which are members of both libraries. Thirty-five targets plus CHEK-1 and PLK-1 had a mean MAD greater than 3 in the three screening experiments and thus were considered as potential hits (Figure 2F). Eighteen of these targets showed a robust phenotype with MAD of greater than 3 in all three screening experiments (Figure 2B), while 24 targets were significant in two of the three experiments (Figure 2C) and 123 targets showed MAD greater than 3 in just one experiment (Figure 2D). The remaining 725 targets showed no response beyond the threshold in any experiment. The categories of targets with one or two experiments with MAD greater than 3 showed the highest standard deviations due to the occurrence of outliers. These categories also contained targets with a small standard deviation, suggesting that their importance for survival of HUVEC was not sufficiently strong for the chosen assay conditions (Figure 2B and C). Every sixth target fell into these categories of unclear importance, thus five of six targets were reproducibly classified as either important or not relevant for survival of HUVEC (Figure 2C and D). Generally, a considerable degree of variation was seen in the data, including the most significant hit category (Figure 2B). The most striking examples were MYC, which nevertheless could be validated, and CHEK1, which was a member of the library and also served as a positive control (Figure 2B and F). The degree of data variation might be attributed to not fully standardized cell culture conditions during the preparation of the experiments and thus argues for our strategy to select the primary hits from repeated screening experiments. As a consequence, we chose all 35 primary hits despite their standard deviation for further validation experiments. While a higher number of primary hits for validation lowered the probability for erroneously classified false negatives, it also lowered the validation rate. Of the 35 identified primary hits, 33 had a pro-proliferative/anti-apoptotic function, as their down-regulation led to increased cell death, while 2 had an anti-proliferate effect as their knockdown allowed for better cell viability. The 16 strongest of the 33 pro-proliferative hits plus the two positive controls were selected for further evaluation (Table 1).

Bottom Line: Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability.Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect.Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

View Article: PubMed Central - PubMed

Affiliation: Lonza Cologne AG, Nattermannallee 1, 50829 Cologne, Germany.

ABSTRACT
Meaningful RNAi-based data for target gene identification are strongly dependent on the use of a biologically relevant cell type and efficient delivery of highly functional siRNA reagents into the selected cell type. Here we report the use of the Amaxa(R) Nucleofector(R) 96-well Shuttle(R) System for siRNA screening in primary cells. Lonza's Clonetics(R) HUVEC-Human Umbilical Vein Endothelial Cells were transfected with Thermo Scientific Dharmacon siGENOME(R) siRNA Libraries targeting protein kinases and cell cycle related genes and screened for genes important for cell viability. Of the 37 primary hits, down-regulation of 33 led to reduced proliferation or increased cell death, while down-regulation of two allowed for better cell viability. The validated four genes out of the 16 strongest primary hits (COPB2, PYCS, CDK4 and MYC) influenced cell proliferation to varying degrees, reflecting differing importance for survival of HUVEC cells. Our results demonstrate that the Nucleofector(R) 96-well Shuttle(R) System allows the delivery of siRNA libraries in cell types previously considered to be difficult to transfect. Thus, identification and validation of gene targets can now be conducted in primary cells, as the selection of cell types is not limited to those accessible by lipid-mediated transfection.

No MeSH data available.