3.

RESULTS AND DISCUSSIONS:

3.1.1. LADDER

FIGURE 2: MOLECULAR LADDER OF SIZE 50bp

This was the molecular ladder used as the reference. The bands till 250 bp have been labeled.

3.2. RESULT AND DISCUSSION FOR FIRST SET OF PCR EXPERIMENT:

3.2.1. RESULT:

FIGURE 3: PAGE IMAGE FOR FIRST SET OF PCR EXPERIMENT.

LANE 1: cDNA prepared from 250 ng of RNA from sample NOA 1
LANE 2: cDNA prepared from 500 ng of RNA from sample NOA 1
LANE 3: cDNA prepared from 250 ng of RNA from sample NOA 2
LANE 4: cDNA prepared from 500 ng of RNA from sample NOA 2
LANE 5: 50 bp Ladder

3.2.2. DISCUSSION:
From the image of the gel, it is clearly seen that there has been no amplification. This could
be due to many reasons. So trouble-shooting was done to understand the reason for this result
and also to rectify the mistake in consequent PCR experiments.
PCR experiments are of sensitive nature. Minute handling errors could lead to no
amplification. The PCR conditions, viz., primer annealing temperature and time duration for
extension per cycle could have been insufficient. This might also lead to no amplification.
But in this case, the chances of handling errors being the reason for no amplification is
higher.
This conclusion was mainly drawn from the fact that the PCR conditions, namely, primer
annealing temperature and time duration for extension were not set randomly. The annealing
temperature was determined from the primers melting temperature ( ) which is based on
the overall nucleotide content of both forward and reverse primer. And the extension time
was calculated with respect to the expected molecular weight of the products and the
efficiency of Taq polymerase.
So, in the next set of PCR experiments, handling was done with extra care so as to avoid
getting a similar result.

3.3. RESULT AND DISCUSSION FOR SECOND SET OF PCR EXPERIMENT:

3.3.1. RESULT:

FIGURE 4: PAGE IMAGE FOR SECOND SET OF PCR EXPERIMENT. FAINT

AMPLIFICATION SEEN IS INDICATED BY BLACK BOX AND PRIMER DIMER
IS INDICATED BY RED BOX
LANE 1: 50bp Ladder
LANE 5: cDNA prepared from 250 ng of RNA from sample NOA 1
LANE 6: cDNA prepared from 500 ng of RNA from sample NOA 1
LANE 7: cDNA prepared from 250 ng of RNA from sample NOA 2
LANE 8: cDNA prepared from 500 ng of RNA from sample NOA 2
3.3.2. DISCUSSION:
From the image, it can be observed that amplification has occurred in one of the templates
only. But even in that template, the amplification is not very high. This is evident from the
very faint band that is seen. But it is nevertheless amplification. The obtained amplicon is of
molecular size approximately 180 base pairs. We can also see primer dimer formation in
lanes 5, 6 and 7 (See The part of FIGURE 4 indicated with red box)
In the first set of PCR experiment, there was no amplification. The probable reasons for
this result were discussed in section 3.1.2. The second set of PCR experiments were carried
out with the same templates of the gene and primers.

But in the second set of PCR experiment, a faint band is seen in lane 8. The cDNA
template used was prepared from 500 ng of RNA from sample NOA 2. We can conclude that
amplification is possible when 500 ng of RNA was used for cDNA preparation. Weak band is
indicative of weaker expression of the gene.
In lanes 5, 6 and 7, there is no amplification seen. But there are bands seen below the 50
base pair band . This is because of primer dimer formation. Primer dimer formation occurs
because the two primers, forward and reverse primers form a complex. There will be no
extension of the template as the primers have annealed with one another. This happens
usually because the primers are complementary to each other.
From the results of second set of PCR experiment, the amount of RNA needed for the
cDNA preparation was standardized. This standardization was essential as only lesser amount
of RNA was available.
3.4. RESULT AND DISCUSSION FOR THIRD SET OF PCR EXPERIMENT:
3.4.1. RESULT:

FIGURE 5: PAGE IMAGE FOR THIRD SET OF PCR EXPERIMENT

LANE 1: 50bp Ladder
LANE 2: cDNA prepared from 500 ng of RNA from sample NOA 3
LANE 3: cDNA prepared from 500 ng of RNA from sample NOA 4
LANE 4: cDNA prepared from 500 ng of RNA from sample NOA 5

3.4.2. DISCUSSION:
From the image, it can be seen that amplification has occurred in all three templates. But the
band is clearer in lane 4, that is, in the cDNA template prepared from 500 ng of RNA from
sample NOA 5. Though all the amplicons obtained are of similar size, there is variation in
gene expression in the individual samples.
With the help of the ladder, we can deduce that the molecular size of the amplicons
obtained is approximately 180 base pairs.
In PAGE image, a very clear band is seen in the product of cDNA template obtained from
sample NOA 5. So, this was used as positive control in for the fourth set of PCR experiments.
A positive control is needed for PCR experiment to know if the experiment has been done
properly. If the experiment was done properly, then the amplification will be seen in positive
control. If amplification is not detected in the positive control, then the experiment has not
been carried out properly. This is the reason why a positive control is used.

3.5. RESULT AND DISCUSSION FOR FOURTH SET OF PCR EXPERIMENT:

3.5.1. RESULT:

FIGURE 6: PAGE IMAGE OF FOURTH SET OF PCR EXPERIMENT

LANE 1: cDNA prepared from 500 ng of RNA from sample NOA 5 (POSITIVE
CONTROL)
LANE 2: cDNA prepared from 500 ng of RNA from sample NOA 6
LANE 3: cDNA prepared from 500 ng of RNA from sample NOA 7
LANE 4: cDNA prepared from 500 ng of RNA from sample NOA 8
LANE 5: cDNA prepared from 500 ng of RNA from sample NOA 9
LANE 6: cDNA prepared from 500 ng of RNA from sample NOA 10
LANE 7: cDNA prepared from 500 ng of RNA from sample NOA 11
LANE 8: cDNA prepared from 500 ng of RNA from sample NOA 12
LANE 9: 50 bp Ladder
3.5.2. DISCUSSION:
It can be clearly seen from the PAGE image that a uniform amplification has been achieved
in all the eight templates. The cDNA template obtained from sample NOA 5 was used as the
positive control. The obtained amplicons are approximately180 base pairs in size.

And from this, we can conclude that the standardization process has been successful. And
the future PCR experiments can be carried out under these standardized conditions. The
standardization of the amount of RNA needed for cDNA preparation was determined to be
500 ng.
3.6. RESULT AND DISCUSSION FOR STANDARDIZATION OF ISOLATION OF
RNA USING TRIZOL METHOD:
3.6.1. RESULT:

FIGURE 7: FAGE IMAGE SHOWING THE ISOLATED RNA FROM 4 BLOOD

SAMPLES
LANE 1: RNA isolated from blood sample 1
LANE 2: RNA isolated from blood sample 2
LANE 3: RNA isolated from blood sample 3
LANE 4: RNA isolated from blood sample 4

3.6.2. DISCUSSION:
From the image, it can be seen that RNA has been isolated from three of the four samples,
namely, sample 1, 3 and 4. It can be observed from the image that, in the lanes 1, 3 and 4,
two bands are seen. These two bands correspond to rRNAs. The upper band indicates 28S
rRNA and the lower band indicates 18S rRNA.
In lane 4, the demarcation between the two bands is not quite distinct. Both the upper
and lower band in lane 4 appears to be a smear. This is usually due to the degradation of the
28S rRNA component.
In lane 3 also, the demarcation between the two bands is not very clear but it is better
than what was seen in lane 4.
As we used different samples, the time taken from the collection of sample to the start
of the protocol varied between the four samples. This might be a reason for obtaining poor
quality RNA from samples 3 and 4.
There are no bands to be observed in lane 2, indicating that RNA isolated has been
degraded. RNA is less stable than DNA. And hence, RNA is more prone to degradation.
In lane 1, both the upper and the lower band are quite distinct.
The quality of the isolated RNA can usually be assessed from the FAGE image. The
better the demarcation between the two bands better is the quality of the RNA isolated. That
implies that the RNA isolated from sample 1 is of higher quality than that from sample 3 and
sample 4.
As we got the bands in three of the four samples used, we can conclude that the
isolation procedure used was correct and the standardization for isolation of RNA was
successful.

4 CONCLUDING REMARKS:
To get proper results from any project, the standardization of the techniques that are used in
the project are very important. That is the main reason why this projects design was divided
into two phases. Though significant results will be produced only at the end of phase-II, the
experiments in phase-I have their own importance as these experiments form the base for the
proper carrying out phase-II experiments.
There were two objectives of standardization of the PCR procedure. One was to
standardize the amount of RNA required to prepare the cDNA template for RT-PCR
experiments. And the second objective was to determine the conditions under which the
RT-PCR is to be carried out. Both these objectives were met at the end of standardization.
Similarly, the standardization of RNA isolation protocol was also met with success.