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Quantitate miRNA from a Few Drops of Mouse Blood
Mouse RiboPure™-Blood RNA Isolation Kit
TaqMan® MicroRNA Assays
While inbred mouse models have been created for most human genetic disorders, including cancer and neurological diseases, blood samples from these mice have not been widely used in biomarker discovery studies. Two major obstacles are ex vivo changes in RNA expression levels introduced during collection, storage, and transport of blood, and the lack of reliable extraction methods with sufficient yields of high-quality RNA. Moreover, mice typically are sacrificed to obtain sufficient blood RNA for analysis, which does not allow for time course studies. This article describes how the Mouse RiboPure™-Blood Kit overcomes all of these issues. This kit can be used to isolate high-quality total RNA, including miRNA, from tail vein bleeds in time course studies. MicroRNA can then be quantitated using TaqMan® MicroRNA Assays.
TaqMan® MicroRNA Assays
While inbred mouse models have been created for most human genetic disorders, including cancer and neurological diseases, blood samples from these mice have not been widely used in biomarker discovery studies. Two major obstacles are ex vivo changes in RNA expression levels introduced during collection, storage, and transport of blood, and the lack of reliable extraction methods with sufficient yields of high-quality RNA. Moreover, mice typically are sacrificed to obtain sufficient blood RNA for analysis, which does not allow for time course studies. This article describes how the Mouse RiboPure™-Blood Kit overcomes all of these issues. This kit can be used to isolate high-quality total RNA, including miRNA, from tail vein bleeds in time course studies. MicroRNA can then be quantitated using TaqMan® MicroRNA Assays.
RNA Isolation
RNA quality can have a significant impact on downstream analyses such as real-time RT-PCR and array analysis. RNA purification from whole blood requires a stringent isolation method that yields high-quality RNA depleted of inhibiting agents and contaminants (e.g., enzymes and anticoagulants).
The Mouse RiboPure-Blood RNA Isolation Kit is designed to recover total RNA—and optionally miRNA—directly from mouse or rat blood in ~35 minutes. The standard sample volume is 0.25–0.5 mL per reaction (e.g., mouse blood collected through cardiac puncture); however, minor procedural modifications included in the Instruction Manual allow smaller volumes to be used (e.g., mouse blood collected from the tail vein or an orbital bleed).
The kit includes microfuge tubes pre-loaded with RNAlater® Tissue Collection:RNA Stabilization Solution, which prevents RNA degradation and preserves RNA profiles within samples at room temperature for up to 3 days or at –20ºC for long term. Typically, 30–55 µg of total RNA (containing miRNA) is obtained from 0.5 mL mouse blood. Yields from rat blood are more variable, generally 18–40 µg from 0.5 mL blood.
The Mouse RiboPure-Blood RNA Isolation Kit is designed to recover total RNA—and optionally miRNA—directly from mouse or rat blood in ~35 minutes. The standard sample volume is 0.25–0.5 mL per reaction (e.g., mouse blood collected through cardiac puncture); however, minor procedural modifications included in the Instruction Manual allow smaller volumes to be used (e.g., mouse blood collected from the tail vein or an orbital bleed).
The kit includes microfuge tubes pre-loaded with RNAlater® Tissue Collection:RNA Stabilization Solution, which prevents RNA degradation and preserves RNA profiles within samples at room temperature for up to 3 days or at –20ºC for long term. Typically, 30–55 µg of total RNA (containing miRNA) is obtained from 0.5 mL mouse blood. Yields from rat blood are more variable, generally 18–40 µg from 0.5 mL blood.
Applications
The resulting RNA can be used in a variety of downstream procedures including real-time RT-PCR and array analysis. For example, real-time RT-PCR was used to monitor changes in miRNA levels in mouse blood over time (Figure 1). The ability to stabilize and isolate high-quality RNA from small blood samples collected from live mice over time makes the Mouse RiboPure-Blood Kit ideal for gene expression studies and time course experiments.
Figure 1. Obtain High Quality RNA Using the Mouse RiboPure™-Blood Kit for Real-Time RT-PCR Analysis of MicroRNA. Blood samples from tail vein bleeds of mice in a time course study were collected in RNA
later® Solution and shipped to Applied Biosystems (Austin, TX). RNA was isolated with the Mouse RiboPure-Blood RNA Isolation Kit. (A) This panel shows a representative electropherogram and gel image (Agilent® 2100 bioanalyzer) from a typical RNA sample. (B) The TaqMan® MicroRNA Reverse Transcription Kit and TaqMan MicroRNA Assays were used to quantify miRNA levels in the samples. These representative results were normalized (?C
T) with results from TaqMan MicroRNA Assays for human RNU6B or mouse snoRNA135. Data is expressed as 40–?C
T, so that there is a positive correlation between the height of the bar and the miRNA expression level. Data courtesy of Drs. AC Fan and DW Felsher, Stanford University, Stanford, CA.