Online and species from which it taken and

Online
databas for northern blotting is BlotBase. Mouse and human northern blots are
published here about 700 in number.there blots are run on 25 types of different
tissues and on 650 genes. This blot can be searched by tissues,blotID,gene
identifier and paper reference.Science community get benefits from this
database and share information.Results on BlotBase give results by providing
gene ,its expression level,its blot ID,tissues and species from which it taken
and blot view(14).

BlotBase:

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Here
is another procedure which is variant of Northern Blotting known as Reverse
Northern Blotting. This procedure we use a fix membrane that contain substrate
consist of small fragments of DNA and radiolabeled probe is RNA, this is
collected from tissues.

Reverse Northern Blotting:

 

·        
In case of northern blotting of
acetylcholinestrase, non-radioactive technique is used. It is considerd more
sensitive as compared to radioactive. It is advantageous as it takes less time.
13

·        
It helps to detect the RNA size, determine the
quality and quantity of RNA on gel before blotting and reprobing of membrane
after blotting.8

·        
It has high specificity. It reduces the risk
of false results.

·        
Northern blot is preffered over microarrays as
it helps to determine very small changes while gene expressing phenomena.12

·        
There are several methods instead of northern
blotting which are quite useful. These include microarrays, RT PCR, RNAase
protection assays as well as SAGE.11

Advantages:

 

·        
It has low sensitivity. 8

·        
Microarrays has advantage over northern
blotting as, by using microarrays, a lot of gene expressions could be read
quickly while northern blot takes time. 9,10

·        
The reagents mostly used in this procedure are
very harmful to the researcher in some circumstances. Chemicals are like
formaldehyde, ethidium bromide, DEPCO and UV light.

·        
The disadvantage of northern blotting is that
it degrades the sample. This sample degradation could be avoided by the use of
RNAase inhibitors i.e DEPCO and glassware sterilization.

Disadvantages:

 

·        
It helps in recombinant screening with the
help of mRNA Obtained by transgene.

·        
Expression patterns help to identify the
function of a particular gene.

·        
It is also used to observe oncogenes as well
as tumor suppressor gene in cancerous cells in contrast with normal cells. 8

·        
It is used to observe gene expressions. In different
fields, helps to observe pattern of gene expression between tissues, organs and
developmental stages. 5,6,7

·        
It is used to detect different diseases as
well as their treatments.

·        
It is used to detect specific mRNA through any
sample.

Applications:

 

To increase the sensitivity as well as detection of highly nonisotropic
blots having low background and high signal/noise ratio make conjugates of
NorthernMax and BRIGHGTSTAR. These conjugates of Northern blots give best results
also with radiolabeled probes.(4)

Different detection kits are available with all material and reagents  for detection of DNA and RNA probes attached
with biotin (biotinylation)  like
BRIGHTSTAR and Nonisotropic Detection kits are used mostly.

Immediately expose to X-ray  film (blot
is enclosed is plastic wrap if it use radiolabel probe to avoid its drying )for
auto radiography. Use nonisotopic reagent for detection of blot which use
nonisotopic probe before its exposure to auto radiography.(3)

Detection:

 

NorthernMax kits have low stringent and high stringent washing buffers
which are certified as RNase free, these washing buffers are available
separately as well.(2)

·        
High stringent washes: Use high strictness in
standards of washing for removal of partially hybridized probes like 0.1X SSC
and SSPE.

·        
Low stringent washes:  Use less strictness in standards of washing
for removal of unhybridized probes like 2X SSC (sodium citrate buffer)and SSPE.

Next step is washing after hybridization , during this step use different
concentrations of buffer to remove unwound or unhybridized probes. Two types of
stringent washes are available:

Washing:

 

All probes are placed in very little quantity of  ULTRAhyb buffer before use ,then finally add
them to the blot.  This step increase
sensitivity by hundred times verses all other hybridization solutions.(1) We
can detect 10,000 molecules minimum by this process because this process is
very fast and can be done in just 2 hours using many messages and as for as
highly sensitive.

Hybridization takes place between the 
DNA and RNA which is he general principle of Northern Blotting.

·        
RNA probes

·        
ss-DNA probe

·        
ds-DNA before use that is
denatured

To avoid problems after probe hybridization we have to do good
prehybridization, this is also known as blocking , coating of the probe on
membrane can be blocked by this step. Coat nonspecific molecules on membrane
where place is not occupied by mRNA.Two most popular kits are used in labs for
both pre-hybridization and hybridization e.g., NorthernMax.Gly and NorthernMax.
Both kits are efficient because they use ultra-sensitive hybridization buffer
(ULTRAhyb). Three types of probes are used in blotting:

Prehybridization and Hybridization with Probe:

 

 For the detection of target RNA
prepare the probes which are identical to the sequence of our target RNA and
mostly present in form of Complementary DNA for Northern Blotting and about minimally
25 base pairs to several thousand nucleotides . Here the most important point
of consideration is that avoid the use of very long probes this can cause
partial binding of probes with non-specific sequences.  We can visit data base searches this will
help and ensure to make the probes which have unique sequences identical to our
target genes. To make the max. hybridization provide large amount of probe so
that it anneals with max. amount of target sequence. If probe is in less
quantity then less hybridization occur and less signal will be detected on the digital
display.

Probe:

x

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