Journal: BMC Genomics

Article Title: DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs

doi: 10.1186/s12864-015-2086-z

Figure Lengend Snippet: Expression of gRNAs. a Primer design for specific detection of gRNAs by qRTPCR. The reverse primer is constant for all PCRs while a specific primer is designed for each gRNA. ( b and c ) Relative expression of transfected gRNAs in HCT116 cells ( b ) and Hela cells ( c ). Cells were transfected and selected with puromycin (2ug/mL) for 3 days. Data are normalised to the housekeeping gene GAPDH. Control amplifications were carried out using indicated primers with cDNA template from pDECKO-GFP cells

Article Snippet: Fig. 3 Expression of gRNAs. a Primer design for specific detection of gRNAs by qRTPCR.

Techniques: Expressing, Transfection, Control

Journal: BMC Genomics

Article Title: DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs

doi: 10.1186/s12864-015-2086-z

Figure Lengend Snippet: Derivation of MALAT1 knockout clones. a MALAT1 RNA expression levels of three HCT116 clones lacking the upstream promoter, deleted using the MALAT1_A DECKO construct. KO, homozygous knockout. Expression detected using Primer Set 1 (see below) and values are relative to a control cell clone expressing pDECKO GFP gRNAs. Error bars indicate the standard deviation of three technical replicates. b As for ( a ) in a heterozygous clone for the major promoter, deleted using the DECKO_C construct. Het, heterozygote. c MALAT1 expression in HEK293T cells heterozygous or homozygous for the major promoter, deleted using the DECKO_C & D constructs. Expression detected using Primer Set 1. d As for ( c ) in HeLa clones, using the DECKO_C & E constructs. e Semi-quantitative PCR carried out on the same HEK293T clones that in Fig. 6c. “RT-“indicates control samples where Reverse Transcriptase was omitted. f - g qRTPCR using three additional downstream primer pairs (see Fig. ), illustrating knockdown of MALAT1 RNA throughout the gene, for the HEK293T and HeLa MALAT1_C clones. h Sequence analysis of mutant junctions. MALAT1_A KO 3 contains one extra C in the deletion point. MALAT1_A 1, MALAT1_A 2 and MALAT1_A 4 have the expected deletion, with no indel. Both gRNA are in antisense direction. Expected cut location is marked with vertical bar. i Proliferation assay comparing the growth rates of control GFP and MALAT1 HEK293T knockout and heterozygous clones. Error bars indicate standard deviation of five cell wells. **, P < 0.01/***, P < 0.001 by Student’s one-sided, paired t test

Article Snippet: Fig. 3 Expression of gRNAs. a Primer design for specific detection of gRNAs by qRTPCR.

Techniques: Knock-Out, Clone Assay, RNA Expression, Construct, Expressing, Control, Standard Deviation, Real-time Polymerase Chain Reaction, Reverse Transcription, Knockdown, Sequencing, Mutagenesis, Proliferation Assay