Biomedical Engineering Reference
In-Depth Information
providing a simple, quick and reliable way to extract high-quality RNA.
However, this technique excludes isolation of small RNA species, such as
rRNA, miRNA, and tRNA (McGarvey
et al.
, 2003).
For isolation of a variety of RNA species, including small RNAs, the
anion-exchange method should be employed. The isolation is carried out by
positively charged resins that selectively bind to RNA while DNA is washed
out for extraction of high-quality RNA (Clontech, 2010; McGarvey
et al.
,
2003).
Determining the quantity and quality of isolated DNA and RNA
After isolation, DNA and RNA samples are recommended to be stored
in TE buffer (10 mM Tris-HCl, 1 mM EDTA) at −20°C (Strauss, 1998;
OpenWetWare, 2009; Applied Biosystems, 2010b). RNA can be stored in an
alternative DEPC-treated, RNase-free water with 0.1 mM EDTA, but TE
buffer is more effective. For long-term storage, both nucleic acid samples
can be stored at −80°C to prevent degradation. Nucleic acid concentrations
can be determined by using a spectrophotometer to measure absorbance
of aqueous nucleic acid samples at 260 nm (A260). Other critical measure-
ments are the ratios of A260/A280 and A260/A230 that indicate the nucleic
acid purity contaminated by, respectively, proteins and organic compounds,
such as phenol. Pure nucleic acids should have an A260/A280 ratio between
1.8-2.0 and an A260/A230 ratio greater than 2.0 (Kisker, 2010), and val-
ues less than these indicate protein or organic compound contamination.
Generally, samples with A260/A280 and A260/A230 ratios greater than 1.8
are used for microarray or quantitative real-time PCR analysis (Sargeant
et al.
, 2010). Importantly, since nucleic acids are easily degraded by nucleases
during sample preparation, it is recommended to examine integrity of RNA
and DNA by gel electrophoresis before subsequent analytical applications.
4.2.2 Quantitative real-time polymerase
chain reaction (PCR)
Quantitative real-time PCR (qPCR) is a versatile and highly sensitive tech-
nique that quantifi es nucleic acids via fl uorescent chemistry. The approach
is designed to produce fl uorescence that is directly related to the amount
of amplifi ed PCR products, thereby allowing researchers to monitor the
amplifi cation profi le during each PCR cycle in a real-time manner. Typical
analyses using real-time PCR include DNA copy number determination
(Konigshoff
et al.
, 2003), single nucleotide polymorphism (SNP) genotyp-
ing (Mhlanga and Malmberg, 2001), DNA damage assessment (Harbottle
et al.
, 2010), and microarray output validation (Chuaqui
et al.
, 2002). The
most common application is to measure relative expression of target genes
