Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A–C) Dose–response curves using double-bubble primer mix #8 and VIC-TqM probe #12 and decimal dilutions of SARS-CoV-2 synthetic RNA N. (A) Real-time amplification plot. C t values: 8 × 10 5 copies, 22.82; 8 × 10 4 copies, 26.27; 8 × 10 3 copies, 30.55; 8 × 10 2 copies, 32.51. (B) 5% agarose gel of the PCR amplification products. Lane 1: ultra-low-range ladder; lanes 2–5: 139-bp amplicons of 8 × 10 5 to 8 × 10 2 RNA copies per tube. Also included in the PCR reaction mix were 2× TqM buffer without UDP and iScript reverse-transcriptase. (C) Efficiency plot of the PCR reaction depicted in panel A. Slope = -3.3359 represents PCR reaction efficiency of 99.42%; R 2 = 0.9816. (D) One-tube RT-qPCR using double-bubble primer mix #10 from region 2 using SARS-CoV-2 synthetic RNA (2 × 10 4 copies per tube) and FAM-TqM probe #13. RT-qPCR was performed with standard conditions.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Amplification, Agarose Gel Electrophoresis, Reverse Transcription, Quantitative RT-PCR

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: (A) qPCR using reverse-transcribed cDNA template and double-bubble (D-B) primer mix #10, FAM-TqM probe #13, TqM fast kit+ uracil-DNA glycosylase (UDG) assayed in duplicates. Insert depicts 5% agarose gel; lane 1: ultra-low-range (ULR) ladder; lanes 2 & 3: template cDNA; lanes 4 & 5: non-template control (NTC). C t average values: cDNA, 28.60; NTC, undetermined. (B) One-tube RT-qPCR using SARS-CoV-2 synthetic RNA N template and D-B primer mix #8, VIC-TqM probe #12, TqM fast kit without UDG iScript reverse transcriptase, assayed in duplicate. Insert depicts 5% agarose gel; lane 1: ULR ladder; lanes 2 and 3: template RNA; lanes 4 and 5: NTC. C t average values: RNA, 24.63, NTC, undetermined. (C) Duplex qPCR using SARS-CoV-2 synthetic RNA N template and TqM fast kit with UDG. C t values: primer 8, 26.13; primer 10, 32.19; duplex primers 8 + 10, 26.36 and 30.27, respectively; NTC, undetermined. (D) 5% agarose gel of samples shown in panel C. Lane 1: ULR ladder; lane 2: primer D-B mix #8 (amplicon 139 bp); lane 3: D-B primer mix #10 (amplicon 158 bp); lane 4: duplex of both primers #8 and #10 (amplicons 139 and 158 bp). Arrows (C & D) show duplex amplifications in the same tube with D-B primers #8 and #10. PCR was performed using fast conditions with 40 (A, C & D) and 30 (B) cycles.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Agarose Gel Electrophoresis, Control, Quantitative RT-PCR, Amplification

Journal: Biotechniques

Article Title: Improved SARS-CoV-2 PCR detection and genotyping with double-bubble primers

doi: 10.2144/btn-2021-0063

Figure Lengend Snippet: The reaction mixture contained hot-start double-bubble (D-B) primer mix #4, VIC-TqM probe #12, cost-effective non-hot-start Taq polymerase (FroggaBio), reverse transcriptase (iScript), synthetic SARS-CoV-2 RNA gene N as template, added dNTPs and ROX dye for internal calibration. The reaction was assembled at room temperature and subjected to real time RT-qPCR using the following fast conditions: 42°C, 15 min; 95°C,1 min; 30 cycles of 95°C, 1 s → 70°C, 20 s. The amplification plot and (insert) 5% agarose gel depict the amplification of SARS-CoV-2 RNA extracted from the nasopharyngeal swabs of patient S1 (lane 2) and negative patient N1 (lane 3), no-template controls (lanes 4 and 5) and SARS-CoV-2 gene N synthetic RNA as positive control (lane 6). Note the amplification in the real-time plot and the 115-bp band in the 5% agarose gel with SARS-CoV-2 virus or synthetic RNA (lanes 2 and 6), with no amplifications in the negative control N1 (lane 3) or NTC (lanes 4 and 5). Lane 1: ultra-low-range ladder. C t values: positive patient S1: 23.95; synthetic RNA: 23.84; negative patient N1 and NTC: undetermined.

Article Snippet: For cDNA synthesis, iScript reverse-transcriptase kit was employed: 25°C, 5 min, 42°C, 15 min 95°C, 1 min, 4°C hold (BioRad, 1708891).

Techniques: Reverse Transcription, Quantitative RT-PCR, Amplification, Agarose Gel Electrophoresis, Positive Control, Virus, Negative Control

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Exploration of PCIF1 target genes by genome-wide gene expression analysis. ( A ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) or two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2). ( B ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2) with anti-PCIF1 and anti-b-actin antibodies. ( C , D ) Venn diagrams showing the overlap between the two indicated siRNA-mediated downregulated ( C ) and upregulated ( D ) genes identified by the gene expression profile analyzed by DNA microarray using a Human Genome U133 Plus 2.0 Array (Affymetrix). The condition for selecting differentially expressed genes are as follows: (1) cut-off condition: expression > 100, (2) fold change: >2, (3) p -value < 0.05.

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Genome Wide, Gene Expression, Quantitative RT-PCR, Isolation, Control, Western Blot, Microarray, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Expression of RAB23 and CNOT6 is regulated by PCIF1 at both the mRNA and protein levels. ( A – C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3), using the specific primer set detecting PCIF1 ( A ), RAB23 ( B ), and CNOT6 ( C ) expression. ( D , E ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) with the indicated antibodies. Signal intensities obtained from immunoblotting were quantified using ImageJ software version 1.52. The y-axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Western Blot, Software, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant PCIF1 restores the normal levels of target mRNA expression. ( A , B ) RT-qPCR analysis of total RNAs isolated from HeLa cells transfected with a control empty vector (Vec) or a vector expressing siRNA-resistant PCIF1 (PCIF1siR) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1 #3), using the specific primer set detecting RAB23 ( A ) and CNOT6 ( B ) expression. The y -axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between indicated pairs (Student’s t -test, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Transfection, Control, Plasmid Preparation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 does not regulate expression of its target genes at the transcriptional level. ( A , E ) Schematic illustrations of the RAB23 ( A ) and CNOT6 ( E ) genes. The transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR primer set for RT-qPCR amplification of the precursor and mature mRNAs are indicated by arrows. ( B – D , F – H ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) using the indicated primer sets. ( I , J ) ChIP analyses of the RAB23 ( I ) and CNOT6 ( J ) gene promoters (position 2 in ) using antibodies against Pol II in HeLa cells treated with control siRNA (siNC) and two distinct PCIF1 targeted siRNAs (siPCIF1 #1 and #3). Normal rabbit IgG was used as the negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1 targeted siRNA (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Isolation, Control, Negative Control, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 regulates the stability of target gene mRNAs in opposite ways. After HeLa cells were treated with a negative control siRNA (siNC) and two PCIF1-targeted siRNAs (siPCIF1 #1 and #3) for 72 h, actinomycin D was added to inhibit transcription. Cells were harvested at 0, 2, 4, 8, and 12 h after treatment, and total RNA was isolated. The amount of residual mRNA was analyzed by RT-qPCR at each time point, using the specific primer set detecting PCIF1 ( A ), CRAB23 ( B ), and CNOT6 ( C ) mRNAs. The relative value was calculated using the expression level of β-actin mRNA (ACTB) as a normalizer. The relative values at each time point were calculated relative to time zero. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Negative Control, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant wild-type but not methyltransferase-deficient mutant PCIF1 restored normal levels of target mRNA expression. ( A ) Immunoblotting analysis of total protein extracts from HeLa cells transfected with a control empty vector (Vec), a vector expressing siRNA-resistant wild-type PCIF1 (siR_wt), or methyltransferase-deficient mutant PCIF1 (siR_mut) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression) with the indicated antibodies. ( B , C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated as in ( A ), using the specific primer set detecting RAB23 ( B ) and CNOT6 ( C ) expression. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Mutagenesis, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 suppression resulted in a significant decrease in m 6 A levels of both RAB23 and CNOT6 mRNAs. MeRIP-qPCR analysis was performed using HeLa cells treated with control siRNA (siNC) or PCIF1-targeted siRNA (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression). RT-qPCR analysis of RNAs purified from the immunoprecipitates of HeLa cell extracts using the anti-m 6 A antibody, using the specific primer set detecting RAB23 ( A ), CNOT6 ( B ), and ACTB ( C ) mRNAs. The y -axis represents the fold change relative to the RNA levels in the immunoprecipitate by anti-m 6 A antibody from HeLa cells treated with control siRNA (siNC). Normal rabbit IgG (IgG) was used as a negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Control, Quantitative RT-PCR, Purification, Negative Control, Standard Deviation

Journal: Nature Communications

Article Title: Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA

doi: 10.1038/s41467-023-43389-7

Figure Lengend Snippet: a Schematic illustration of the conventional CRISPR-Cas12a assay by a single full-sized crRNA. b Working principle of the asymmetric CRISPR assay using two competitive crRNAs (full-sized crRNA and split crRNA). The full-sized crRNA has a higher affinity to Cas12a and specifically binds the target nucleic acid. The split crRNA is designed to bind to its own ssDNA sequence (split-T), which is different from the target nucleic acid. Due to the different binding affinities of the two crRNAs with CRISPR, CRISPR-Cas12a is first activated by the full-sized crRNA and its target nucleic acid and then is reactivated by the split crRNA and its split-T, resulting in cascade signal amplification of nucleic acid detection. N.C., negative control. Illustrations were created with BioRender.com.

Article Snippet: Next, the RNA solution was mixed with the asymmetric CRISPR assay reaction components including 1X NEBuffer 2.1 buffer, 100 nM LbCas12a, 40 nM crRNA, 10 nM each split crRNA (handle and spacer), 20 nM DNA activator, and 1 μM fluorescence-quencher probe.

Techniques: CRISPR, Sequencing, Binding Assay, Amplification, Negative Control

Journal: Nature Communications

Article Title: Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA

doi: 10.1038/s41467-023-43389-7

Figure Lengend Snippet: a Schematic illustration of miRNA detection based on the asymmetric CRISPR assay. Illustration was created with BioRender.com. b ΔFluorescence intensity (F Target miRNA – F control ) depending on the full-sized crRNA concentration from 10 to 60 nM. [Cas12a] = 100 nM, [split crRNA] = 10 nM, [DNA activator] = 20 nM. ( n = 3, Data are represented as mean ± S.D of three technical replicates). c ΔFluorescence intensity depending on the split crRNA concentration from 10 to 80 nM. [Cas12a] = 100 nM, [RD crRNA] = 40 nM, [DNA activator] = 20 nM. ( n = 3, Data are represented as mean ± S.D of three technical replicates). d ΔFluorescence intensity depending on the DNA activator concentration from 10 to 80 nM. [Cas12a] = 100 nM, [split crRNA] = 10 nM, [RD crRNA] = 40 nM. ( n = 3, Data are represented as mean ± S.D of three technical replicates). e ΔFluorescence intensity depending on the Cas12a concentration from 25 to 200 nM. [split crRNA] = 10 nM, [RD crRNA] = 40 nM, [DNA activator] = 20 nM. ( n = 3, Data are represented as mean ± S.D of three technical replicates) miR-19a sequence was used as a target, and the concentration was 10 pM. f Time-dependent fluorescence signal changes of the asymmetric CRISPR assay (red) and Cas12a/crRNA reaction (black). Inset is the Δfluorescence intensity (F Target miRNA – F control ) comparison of the asymmetric CRISPR assay (red) and Cas12a/crRNA reaction (black). The target miR-19a concentration is 1 pM. Each curve was subtracted by the control signal. ( n = 7 and data represent mean ± S.D of seven technical replicates). g ΔFluorescence intensity of the asymmetric CRISPR assay (red) and Cas12a/crRNA (black) as a function of the target miRNA concentrations. Inset is the linear relationship between the fluorescence signal and logarithm concentration of miR-19a ranging from 1 fM to 10 pM. ( n = 7 and data represent mean ± S.D of seven technical replicates). h , i Time-dependent fluorescence signal and Δfluorescence intensity of different types of miRNAs (miR-19a, let-7a, miR-21, miR-155, and miR-122). [miR-19a] = 100 pM, [let-7a, miR-21, miR-155, and miR-122] = 1 nM. ( n = 3, Data are represented as mean ± S.D of three technical replicates) Source data are provided as a Source Data file.

Article Snippet: Next, the RNA solution was mixed with the asymmetric CRISPR assay reaction components including 1X NEBuffer 2.1 buffer, 100 nM LbCas12a, 40 nM crRNA, 10 nM each split crRNA (handle and spacer), 20 nM DNA activator, and 1 μM fluorescence-quencher probe.

Techniques: CRISPR, Control, Concentration Assay, Sequencing, Fluorescence, Comparison

Journal: Nature Communications

Article Title: Asymmetric CRISPR enabling cascade signal amplification for nucleic acid detection by competitive crRNA

doi: 10.1038/s41467-023-43389-7

Figure Lengend Snippet: a Schematic illustration of miRNA detection in human plasma samples using the asymmetric CRISPR assay. Illustration was created with BioRender.com. b Estimated target miR-19a concentrations in the bladder patient plasma samples (samples 1–10) and the healthy donor plasma samples (samples 11–15). The Blue dashed line represents the highest target miRNA concentration of healthy donors plus standard deviation. ( n = 3, Data are represented as mean ± S.D of three technical replicates.) c Heatmap of the estimated target miR-19a concentrations of the asymmetric CRISPR assay and conventional RT-qPCR assay. d miR-19a expression level analyzed by asymmetric CRISPR (red) and RT-qPCR (black) in bladder patients and healthy donors. The center line represents the median expression level, the bounds of the box indicate the interquartile range, and the whiskers indicate the maximum and minimum values. Source data are provided as a Source Data file.

Article Snippet: Next, the RNA solution was mixed with the asymmetric CRISPR assay reaction components including 1X NEBuffer 2.1 buffer, 100 nM LbCas12a, 40 nM crRNA, 10 nM each split crRNA (handle and spacer), 20 nM DNA activator, and 1 μM fluorescence-quencher probe.

Techniques: CRISPR, Concentration Assay, Standard Deviation, Quantitative RT-PCR, Expressing