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Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

Boos JA, Kirk DW, Piccolotto ML, Zuercher W, Gfeller S, Neuner P, Dattler A, Wishart WL, Von Arx F, Beverly M, Christensen J, Litherland K, van de Kerkhof E, Swart PJ, Faller T, Beyerbach A, Morrissey D, Hunziker J, Beuvink I - Nucleic Acids Res. (2013)

Bottom Line: Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides.Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image.Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research (NIBR), Novartis Pharma AG, Basel, Basel-Stadt CH-4056, Switzerland and NIBR, Novartis Pharma AG, Cambridge, Massachusetts, MA 02139, USA.

ABSTRACT
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

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Characterization and validation of the miRNA RT-qPCR method. (a) RT-qPCR assay for miRNAs and siRNAs. The RT/Reverse primer is used to generate a cDNA copy of the RNA template during the Reverse Transcription step. Real-time fluorescent quantification occurs during the qPCR step with the help of a fluorescently labelled Forward primer hybridizing to the elongated RT/Reverse primer. Signals generated by non-incorporated forward primers are quenched by a quencher-labelled anti-primer. (b) The specificity of the RT-qPCR method was verified by analysing the back-ground signal intensity generated by let-7 miRNA-specific primers using either Let-7a, -7b, -7c, -7d, -7e, -7f, -7g, -7i as template in the reaction. Average values for the perfectly matched targets were set at 100%. Error bars, STDEV (n = 4). (c) miRNA expression profile in diluted tissue homogenates: Thymus (Th), Lung (Lu), Heart (H), Skeletal Muscle (S.M), Kidney (K), Brain (B), Liver (Li) and Spleen (S). Signals were normalized against genomic 18S, and the maximum averaged signal for each mRNA was set to 100%.
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gkt515-F2: Characterization and validation of the miRNA RT-qPCR method. (a) RT-qPCR assay for miRNAs and siRNAs. The RT/Reverse primer is used to generate a cDNA copy of the RNA template during the Reverse Transcription step. Real-time fluorescent quantification occurs during the qPCR step with the help of a fluorescently labelled Forward primer hybridizing to the elongated RT/Reverse primer. Signals generated by non-incorporated forward primers are quenched by a quencher-labelled anti-primer. (b) The specificity of the RT-qPCR method was verified by analysing the back-ground signal intensity generated by let-7 miRNA-specific primers using either Let-7a, -7b, -7c, -7d, -7e, -7f, -7g, -7i as template in the reaction. Average values for the perfectly matched targets were set at 100%. Error bars, STDEV (n = 4). (c) miRNA expression profile in diluted tissue homogenates: Thymus (Th), Lung (Lu), Heart (H), Skeletal Muscle (S.M), Kidney (K), Brain (B), Liver (Li) and Spleen (S). Signals were normalized against genomic 18S, and the maximum averaged signal for each mRNA was set to 100%.

Mentions: The RNA biodistribution data reported above were obtained using the highly specific TaqMan assay, which relies on the specific amplification of a target gene using two primers and the hydrolysis of a TaqMan probe. Although similar probe-based assays are described for the detection of siRNAs (22,23), we set out to develop an RT-qPCR assay with the aim of reducing the amount of sample handling steps without compromising assay specificity or sensitivity (Figure 2a). Like most assays, the first step relies on the classical conversion of RNA into cDNA by reverse transcriptase-mediated primer extension (24). In the second step, a qPCR-mix is directly added to the heat-inactivated reverse-transcription reaction followed by an anti-primer quenching-based real-time PCR reaction (25). Real-time fluorescent quantification takes place during the extension cycle of the PCR reaction. During this cycle, the un-incorporated forward primers will be quenched by the quencher-labelled anti-primer whereas forward primers that constitute the double-stranded PCR products will not. An exponentially increasing fluorescent signal will be the result of successful target amplification. Because miRNAs and siRNAs are similar in size and hence pose similar challenges with respect to designing reliable primers, the specificity of the assay was evaluated in a typical matrix study whereby the level of cross-reactivity was measured against a panel of closely related Let-7 miRNA family members (Figure 2b). Taken into account that the perfectly matched target was arbitrarily set at 100%, the highest cross-reactivity (∼3%) was observed only when Let-7d and Let-7b levels were determined using primers designed against Let-7a or Let-7c, respectively. All other template/primer combinations resulted in minimal cross-reactivity ranging between 0 and 1%. Similar levels of specificity were reported using commercially available TaqMan miRNA assays (26). In addition to this, all the Let-7 assays displayed a large dynamic range (8–10 logs) with a lower limit of detection ranging from 0.02 to 0.002 femtogram, highlighting the level of sensitivity that can be achieved using this RT-qPCR assay (Supplementary Figure S3). The performance of the RT-qPCR assay was further characterized by measuring the relative expression levels of a panel of miRNAs in the same mouse organ homogenates described previously (Figure 2c and Supplementary Table S3). As expected, detection of the well characterized heart- and muscle-specific miRNAs (e.g. miR-208a, miR-1a, miR-133a), liver-specific (e.g. miR-122) and brain-enriched miRNAs (e.g. miR-124, miR-127, miR-128, miR-132, miR-137, miR-139) in the appropriate organs (27–29) confirms the compatibility of our method with measuring miRNAs directly in tissue lysates. In addition to this, RT-qPCR analysis of miRNA 122-5p, 208a-3p, 124-3p, 124-5p, 191-5p and 16-5p using total RNA as input in the reaction resulted in similar expression profiles (Supplementary Figure S4 and Supplementary Table S4). Taken together, the data strongly suggest that the lysis procedure is indeed compatible with the specific detection of tissue-derived miRNAs.Figure 2.


Whole-body scanning PCR; a highly sensitive method to study the biodistribution of mRNAs, noncoding RNAs and therapeutic oligonucleotides.

Boos JA, Kirk DW, Piccolotto ML, Zuercher W, Gfeller S, Neuner P, Dattler A, Wishart WL, Von Arx F, Beverly M, Christensen J, Litherland K, van de Kerkhof E, Swart PJ, Faller T, Beyerbach A, Morrissey D, Hunziker J, Beuvink I - Nucleic Acids Res. (2013)

Characterization and validation of the miRNA RT-qPCR method. (a) RT-qPCR assay for miRNAs and siRNAs. The RT/Reverse primer is used to generate a cDNA copy of the RNA template during the Reverse Transcription step. Real-time fluorescent quantification occurs during the qPCR step with the help of a fluorescently labelled Forward primer hybridizing to the elongated RT/Reverse primer. Signals generated by non-incorporated forward primers are quenched by a quencher-labelled anti-primer. (b) The specificity of the RT-qPCR method was verified by analysing the back-ground signal intensity generated by let-7 miRNA-specific primers using either Let-7a, -7b, -7c, -7d, -7e, -7f, -7g, -7i as template in the reaction. Average values for the perfectly matched targets were set at 100%. Error bars, STDEV (n = 4). (c) miRNA expression profile in diluted tissue homogenates: Thymus (Th), Lung (Lu), Heart (H), Skeletal Muscle (S.M), Kidney (K), Brain (B), Liver (Li) and Spleen (S). Signals were normalized against genomic 18S, and the maximum averaged signal for each mRNA was set to 100%.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
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gkt515-F2: Characterization and validation of the miRNA RT-qPCR method. (a) RT-qPCR assay for miRNAs and siRNAs. The RT/Reverse primer is used to generate a cDNA copy of the RNA template during the Reverse Transcription step. Real-time fluorescent quantification occurs during the qPCR step with the help of a fluorescently labelled Forward primer hybridizing to the elongated RT/Reverse primer. Signals generated by non-incorporated forward primers are quenched by a quencher-labelled anti-primer. (b) The specificity of the RT-qPCR method was verified by analysing the back-ground signal intensity generated by let-7 miRNA-specific primers using either Let-7a, -7b, -7c, -7d, -7e, -7f, -7g, -7i as template in the reaction. Average values for the perfectly matched targets were set at 100%. Error bars, STDEV (n = 4). (c) miRNA expression profile in diluted tissue homogenates: Thymus (Th), Lung (Lu), Heart (H), Skeletal Muscle (S.M), Kidney (K), Brain (B), Liver (Li) and Spleen (S). Signals were normalized against genomic 18S, and the maximum averaged signal for each mRNA was set to 100%.
Mentions: The RNA biodistribution data reported above were obtained using the highly specific TaqMan assay, which relies on the specific amplification of a target gene using two primers and the hydrolysis of a TaqMan probe. Although similar probe-based assays are described for the detection of siRNAs (22,23), we set out to develop an RT-qPCR assay with the aim of reducing the amount of sample handling steps without compromising assay specificity or sensitivity (Figure 2a). Like most assays, the first step relies on the classical conversion of RNA into cDNA by reverse transcriptase-mediated primer extension (24). In the second step, a qPCR-mix is directly added to the heat-inactivated reverse-transcription reaction followed by an anti-primer quenching-based real-time PCR reaction (25). Real-time fluorescent quantification takes place during the extension cycle of the PCR reaction. During this cycle, the un-incorporated forward primers will be quenched by the quencher-labelled anti-primer whereas forward primers that constitute the double-stranded PCR products will not. An exponentially increasing fluorescent signal will be the result of successful target amplification. Because miRNAs and siRNAs are similar in size and hence pose similar challenges with respect to designing reliable primers, the specificity of the assay was evaluated in a typical matrix study whereby the level of cross-reactivity was measured against a panel of closely related Let-7 miRNA family members (Figure 2b). Taken into account that the perfectly matched target was arbitrarily set at 100%, the highest cross-reactivity (∼3%) was observed only when Let-7d and Let-7b levels were determined using primers designed against Let-7a or Let-7c, respectively. All other template/primer combinations resulted in minimal cross-reactivity ranging between 0 and 1%. Similar levels of specificity were reported using commercially available TaqMan miRNA assays (26). In addition to this, all the Let-7 assays displayed a large dynamic range (8–10 logs) with a lower limit of detection ranging from 0.02 to 0.002 femtogram, highlighting the level of sensitivity that can be achieved using this RT-qPCR assay (Supplementary Figure S3). The performance of the RT-qPCR assay was further characterized by measuring the relative expression levels of a panel of miRNAs in the same mouse organ homogenates described previously (Figure 2c and Supplementary Table S3). As expected, detection of the well characterized heart- and muscle-specific miRNAs (e.g. miR-208a, miR-1a, miR-133a), liver-specific (e.g. miR-122) and brain-enriched miRNAs (e.g. miR-124, miR-127, miR-128, miR-132, miR-137, miR-139) in the appropriate organs (27–29) confirms the compatibility of our method with measuring miRNAs directly in tissue lysates. In addition to this, RT-qPCR analysis of miRNA 122-5p, 208a-3p, 124-3p, 124-5p, 191-5p and 16-5p using total RNA as input in the reaction resulted in similar expression profiles (Supplementary Figure S4 and Supplementary Table S4). Taken together, the data strongly suggest that the lysis procedure is indeed compatible with the specific detection of tissue-derived miRNAs.Figure 2.

Bottom Line: Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides.Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image.Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

View Article: PubMed Central - PubMed

Affiliation: Novartis Institutes for Biomedical Research (NIBR), Novartis Pharma AG, Basel, Basel-Stadt CH-4056, Switzerland and NIBR, Novartis Pharma AG, Cambridge, Massachusetts, MA 02139, USA.

ABSTRACT
Efficient tissue-specific delivery is a crucial factor in the successful development of therapeutic oligonucleotides. Screening for novel delivery methods with unique tissue-homing properties requires a rapid, sensitive, flexible and unbiased technique able to visualize the in vivo biodistribution of these oligonucleotides. Here, we present whole body scanning PCR, a platform that relies on the local extraction of tissues from a mouse whole body section followed by the conversion of target-specific qPCR signals into an image. This platform was designed to be compatible with a novel RT-qPCR assay for the detection of siRNAs and with an assay suitable for the detection of heavily chemically modified oligonucleotides, which we termed Chemical-Ligation qPCR (CL-qPCR). In addition to this, the platform can also be used to investigate the global expression of endogenous mRNAs and non-coding RNAs. Incorporation of other detection systems, such as aptamers, could even further expand the use of this technology.

Show MeSH