**7. Methods of MicroRNA analysis and quantification**

Numerous approaches have been developed to analyze and quantify the expression of miRNAs. A commonly adopted strategy is to perform mass scale expression profiling/signature of miRNAs on a small cohort of patients to identify most significantly dysregulated miRNAs. Expression profiling is usually followed by a validation of selected miRNAs on an independent cohort by using QRT-PCR. Expression profiling has been performed using Hybridization-Microarray, Real Time Polymerase Chain Reaction (QRT-PCR) Array and most recently Deep-Sequencing (Meyer, et al, 2010). Most of these approaches are developed against the gold standard 'Northern Blotting'. Each has its unique advantages and disadvantages, such as throughput, sensitivity, ease of use and cost. QRT-PCR can detect very low concentrations of molecules with much superior sensitivity and expenditure of time and money (Chen, et al, 2005). Microarray-based techniques have the advantage of being relatively cost-effective, quick and simple to utilize (Pradervand, et al, 2010). Ultra high throughput miRNA sequencing allows denovo detection and relative quantification of miRNAs, but requires a considerable amount of time and cost for data generation and data analysis (Wang, et al, 2007). A key issue of miRNA detection and quantification is the selection of endogenous controls for relative quantification. In QRT-PCR based detection systems, several small nuclear and small nucleolar RNAs (e.g. RNU6B) are recommended for normalising miRNA expression signature/profiles in tissues, cell lines, and human body fluids. However, RNU6B is heat unstable and rapidly degrades resulting in poor reproducibility of experiments. That's why many researchers have used the invariant and most stable miRNAs as endogenous controls (Meyer, et al, 2010). In order to overcome this problem of normalization in QRT-PCR and other detection systems, researchers have used different statistical strategies including: global mean expression; quantile; scaling; and normalizing factor. However, some normalization methods have been challenged whereas others were adapted to the specific nature of miRNA profiling experiments. At present, there is no generally agreed normalization strategy for any of the known detection approaches. Table 2 shows the comparison of different detection systems by practical application, throughput, cost and time expenditure.

MicroRNAs are Novel Biomarkers for Detection of Colorectal Cancer 7

Bandrés. et al, 2006 miR-133b, miR-145 **miR-31**, miR-96, miR-135b, miR-

Lanza, et al, 2007 miR-17-5p, miR-20, miR-25, miR-

Rossi, et al, 2007 miR-200b, miR-210 , miR-224 miR-19a, miR-20, miR-21, miR-23a,

**Upregulated miRNAs in CRC** 

**miR-16**, miR-17-5p, miR-20a, miR-21, miR-29b ,miR-141, **miR-**

miR-10a, miR-15b ,miR-23a, miR-25, miR-27a, miR-27b, **miR-30c**, miR-107, **miR-125a**, miR-191,

**let-7 g**, miR-181b, miR-200c

miR-25, miR-27a, miR-27b, miR-29a, miR-30e, miR-124b, **miR-132**, **miR-133a**, miR-135b, miR-141, miR-147, miR-151, miR-152, miR-

106a, miR-107, miR-191, miR-221

miR-20a, miR-92, miR-510, miR-

miR-20a, miR-21, **miR-106a**, **miR-**

miR-17-5p, miR-18a, miR-19a, miR-19b, miR-20a, miR-21, miR-25, miR-29a, miR-29b, **miR-31**, miR-34a, miR-93, miR-95, miR-96, **miR-106a**, miR-106b, miR-130b, **miR-181b**, miR-182, miR-183, miR-203, **miR-224**

miR-21, miR-21\*, miR-183, miR-92a, miR-17, miR-18a, miR-19a,

92, miR-93-1, miR-106a

182, miR-185

**181b**, miR-203

miR-34a

miR-145 miR-17-5p ,miR-21, **miR-30c**, miR-

513

**tissue** 

183

**195**, miR-199a

miR-200c, miR-339

**Studies Downredulated miRNAs in CRC tissue** 

Volinia, et al, 2006 let-7a-1, miR-9-3, miR-23b, miR-138, miR-218

Xi, et al, 2006 let-7b, **let-7 g** , miR-26a , miR-

miR-372

Akao, et al, 2006 miR-143, miR-145, let -7

Nakajima, et al,

Monzo, et al,

Schepeler, et al,

Schetter, et al,

2008

2008

2008

Table 3.

2006

30a-3p, **miR-132**, miR-181a, **miR-181b**, miR-296, miR-320,

Slaby, et al, 2007 **miR-31**, miR-143, miR-145 miR-21

miR-484

Arndt, et al, 2009 miR-1, miR-10b, miR-30a-3p,

Slattery, et al, 2011 miR-143, miR-145, miR-192, miR-215

miR-101, miR-145, miR-455,

miR-30a-5p, **miR-30c**, **miR-125a**, **miR-133a**, miR-139, miR-143, miR-145, **miR-195**, miR-378\*, miR-422a, miR-422b, miR-497


Table 2.
