30
3.7 Stable Isotope Probing (SIP)
Stable isotope probing (SIP) is a common and suitable tool to establish appropriate
connection between the structure and function of microorganism communities
when it is linked with the metagenomics (Chen and Murrell 2010). Using this technique we can identify the microbial community in an environmental sample (Chen
and Murrell 2010; Dumont and Murrell 2005) with biomarkers such as DNA and
RNA. The technique is based on the labelling of growth substrate with stable isotopes such as
13
C, which is further incorporated in the cells of microorganisms as
phospholipids, DNA, or RNA. Further, these SIP-tagged nucleic acids and phospholipids can be easily identified against the unlabelled background by using densitydependent centrifugation. DNA and rRNA are the biomarkers that give an idea
about microbial diversity. DNA-SIP, PLFA-SIP, and RNA-SIP are the three major
techniques reported so far. DNA-SIP involves amplifying the isolated DNA through
PCR, then sequencing the 16S rRNA gene and further analysing the phylogeny to
decipher functions of microorganisms along with their community structure
(Dumont and Murrell 2005). The other type of SIP, called PLFA-SIP, has also been
used in the study of microbial identity along with metabolic profiling in the environmental sample (Chen et al. 2008). The sensitivity of PLFA-SIP is much higher than
the DNA- or RNA-mediated SIP (Neufeld et al. 2007). On the basis of PLFA-SIP
profile, it is possible to mark most of the diversity inhabiting the habitat (Taylor
et al. 2013). RNA-SIP is a beneficial technique to understand the ongoing microbebased processes in the environmental samples. The procedure involves isolation of
total RNA followed by a density-dependent centrifugation with 16S rRNA RT-PCR
amplification. Further, DGGE profiling of the PCR products gives a detailed picture
of major microbial communities involved in environmental processes (Manefield
et al. 2002). RNA-SIP is more responsive than DNA-SIP but has major limitations
related to the instability of the mRNA.
3.8 Quantitative PCR (Q-PCR)
Quantitative real-time PCR (qPCR) has been used extensively as a tool for identifying microorganisms of interest and their gene expression from different environmental samples (Higuchi et al. 1992). Real-time PCR provides appropriate
quantification of targeted nucleic acids from even a low amount of the complex
starting material. Moreover, Q-PCR is a versatile tool for highly precise, extremely
responsive, and high-throughput identification and quantification of targeted nucleic
acid sequence from samples of diverse environmental compartments (Sanzani et al.
2014).
Application of Q-PCR strengthened our understanding about the abundance and
role of microbial diversity under various ecosystems. Studies showed a direct link
between the abundance of the specific compositional soil bacterial community and
3 Methods for Exploring Soil Microbial Diversity
3.7 Stable Isotope Probing (SIP)
Stable isotope probing (SIP) is a common and suitable tool to establish appropriate
connection between the structure and function of microorganism communities
when it is linked with the metagenomics (Chen and Murrell 2010). Using this technique we can identify the microbial community in an environmental sample (Chen
and Murrell 2010; Dumont and Murrell 2005) with biomarkers such as DNA and
RNA. The technique is based on the labelling of growth substrate with stable isotopes such as
13
C, which is further incorporated in the cells of microorganisms as
phospholipids, DNA, or RNA. Further, these SIP-tagged nucleic acids and phospholipids can be easily identified against the unlabelled background by using densitydependent centrifugation. DNA and rRNA are the biomarkers that give an idea
about microbial diversity. DNA-SIP, PLFA-SIP, and RNA-SIP are the three major
techniques reported so far. DNA-SIP involves amplifying the isolated DNA through
PCR, then sequencing the 16S rRNA gene and further analysing the phylogeny to
decipher functions of microorganisms along with their community structure
(Dumont and Murrell 2005). The other type of SIP, called PLFA-SIP, has also been
used in the study of microbial identity along with metabolic profiling in the environmental sample (Chen et al. 2008). The sensitivity of PLFA-SIP is much higher than
the DNA- or RNA-mediated SIP (Neufeld et al. 2007). On the basis of PLFA-SIP
profile, it is possible to mark most of the diversity inhabiting the habitat (Taylor
et al. 2013). RNA-SIP is a beneficial technique to understand the ongoing microbebased processes in the environmental samples. The procedure involves isolation of
total RNA followed by a density-dependent centrifugation with 16S rRNA RT-PCR
amplification. Further, DGGE profiling of the PCR products gives a detailed picture
of major microbial communities involved in environmental processes (Manefield
et al. 2002). RNA-SIP is more responsive than DNA-SIP but has major limitations
related to the instability of the mRNA.
3.8 Quantitative PCR (Q-PCR)
Quantitative real-time PCR (qPCR) has been used extensively as a tool for identifying microorganisms of interest and their gene expression from different environmental samples (Higuchi et al. 1992). Real-time PCR provides appropriate
quantification of targeted nucleic acids from even a low amount of the complex
starting material. Moreover, Q-PCR is a versatile tool for highly precise, extremely
responsive, and high-throughput identification and quantification of targeted nucleic
acid sequence from samples of diverse environmental compartments (Sanzani et al.
2014).
Application of Q-PCR strengthened our understanding about the abundance and
role of microbial diversity under various ecosystems. Studies showed a direct link
between the abundance of the specific compositional soil bacterial community and
3 Methods for Exploring Soil Microbial Diversity