What is PCR ? What is Real Time PCR ? Polymerase Chain Reaction ? What is Reverse Transcriptase Enzyme ?
Presented By:
Bharat Bhushan Negi
M.Tech. Biotechnology
IIT Guwahati
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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What is PCR ? What is Real Time PCR ? Polymerase Chain Reaction ? What is Reverse Transcriptase Enzyme ?
Presented By:
Bharat Bhushan Negi
M.Tech. Biotechnology
IIT Guwahati
A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional PCR.
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Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
This is a presentation slide about cellular RNA interference process and RNA interference technology. Contains basic information about biology of cellular RNA interference processes and its discovery, and RNA interference technology. Also gives you the history and development of in-vitro and in-vivo technologies for applicability of RNA interference technology.
siRNA synthesis, siRNA libraries, siRNA delivering techniques, Electroporation, viral transfection methods, Advantages and disadvantages of RNA interference technology.
details about the preliminary and pre-clinical experiments of RNA interference as well as clinical trials of RNA interference.
Arabinose Operon is a self-regulatory sequence of genes used by material to metabolize a five-carbon sugar called arabinose when there is a deficiency of glucose in the environment.
In this slide contains principle, types, methods and application of Western Blotting Technique.
Presented by: T.NIRANJAN REDDY (Department of pharmacology).
RIPER, anantapur
This is a presentation slide about cellular RNA interference process and RNA interference technology. Contains basic information about biology of cellular RNA interference processes and its discovery, and RNA interference technology. Also gives you the history and development of in-vitro and in-vivo technologies for applicability of RNA interference technology.
siRNA synthesis, siRNA libraries, siRNA delivering techniques, Electroporation, viral transfection methods, Advantages and disadvantages of RNA interference technology.
details about the preliminary and pre-clinical experiments of RNA interference as well as clinical trials of RNA interference.
Enabling RNA-Seq With Limited RNA Using Whole Transcriptome AmplificationQIAGEN
RNA-Seq was developed to perform transcriptome profiling and provides a highly precise measurement of expression levels of transcripts and their isoforms. Normally, RNA-Seq analysis requires at least 500 ng –1 μg of total RNA. When working with small biopsies, single cells (such as circulating tumor cells), or other limited material, whole transcriptome amplification (WTA) is normally required. Various WTA methods overcome limited RNA availability and enable transcriptome analysis from limited material or even single cells. In standard PCR-based WTA procedures, however, bias from uneven coverage of cDNA regions with high GC or AT content or amplification errors can lead to the loss of transcripts and wrong variant calling. Here, we compare a standard RNA-Seq library preparation method and the REPLI-g RNA library protocol. The REPLI-g procedure is a PCR-free protocol to efficiently generate RNA-Seq libraries from small amounts of RNA or a single cell in 6.5–7 hours. The REPLI-g protocol uses whole transcriptome amplification based on multiple displacement amplification (MDA), combined with an efficient library adaptor ligation procedure, to prepare RNA-Seq libraries from small RNA amounts. The procedure demonstrates high fidelity, minimal bias and retention of sample‘s transcriptional profile. Compared to standard RNA-Seq library prep, the REPLI-g protocol demonstrates similar reproducibility and sensitivity in transcript detection.
Hotspot mutation and fusion transcript detection from the same non-small cell...Thermo Fisher Scientific
The presence of certain chromosomal Header
rearrangements and the subsequent fusion
gene derived from translocations has been
implicated in a number of cancers. Hundreds of
translocations have been described in the
literature recently but the need to efficiently
detect and further characterize these
chromosomal translocations is growing
exponentially. The two main methods to identify
and monitor translocations, fluorescent in situ
hybridization (FISH) and comparative genomic
hybridization (CGH) are challenging, labor
intensive, the information obtained is limited,
and sensitivity is rather low. Common sample
types for these analyses are biopsies or small
tumors, which are very limited in material
making the downstream measurement of more
than one analyte rather difficult; obtaining
another biopsy, using a different section or
splitting the sample can raise issues of tumor
heterogeneity. The ability to study mutation
status as well as measuring fusion transcript
expression from the same sample is powerful
because you’re maximizing the information
obtained from a single precious sample and
eliminating any sample to sample variation.
Here we describe the efficient isolation of two
valuable analytes, RNA and DNA, from the
same starting sample without splitting, followed
by versatile and informative downstream
analysis. This methodology has been applied to
FFPE and degraded samples as well as fresh
tissues, cells and blood. DNA and RNA were
recovered from the same non-small cell lung
adenocarcinoma sample and both mutation
analysis, as well as fusion transcript detection
was performed using the Ion Torrent PGM™
platform on the same Ion 318™ chip. Using
10ng of DNA and 10ng of RNA input, we
applied the Ion AmpliSeq™ Colon and Lung
Cancer panel to analyze over 500 COSMIC
mutations in 22 genes and the Ion AmpliSeq™
RNA Lung Fusion panel to detect 40 different
fusion transcripts.
RNA analysis on non-denaturing agarose gel electrophoresis.pdffateh11
use 1X TAE buffer instead of 1X TBE
- use agarose gel in the concentration of 1.1%-1.2%
- add ethidium bromide (EtBr) to the gel and electrophoresis buffer to avoid the additional (potentially
RNAse-prone) step of gel staining
- always use fresh gel and buffer as well as clean electrophoresis equipment for RNA analysis. Wear
gloves to protect RNA samples from degradation by nucleases and avoid a hand contact with EtBr. - use
running voltage up to 10 V/cm (10V per each cm of space between the electrodes in electrophoretic
chamber). Do not use high voltage to avoid RNA degradation during electrophoresis.
2. Heat an aliquot of the RNA solution at 70°C for 1 min and place it on ice before loading on a gel.
3. Load a known amount of DNA or RNA ladder alongside your RNA sample as a standard for
determining the RNA concentration. RNA concentration can be roughly estimated assuming that the
efficiency of EtBr incorporation in rRNA is the same as for DNA (the ribosomal RNA may be considered
a double-stranded molecule due to its extensive secondary structure).
4. The first sign of RNA degradation on the non-denaturing gel is a slight smear starting from the rRNA
bands and extending to the area of shorter fragments. RNA showing this extent of degradation is still
good for further procedures. However, if the downward smearing is so pronounced that the rRNA bands
do not have a discernible lower edge, this RNA should be discarded.
The following characteristics indicate successful RNA preparation:
- For mammalian total RNA, two intensive bands should be observed against a light smear. These bands
represent 28S and 18S rRNA. The ratio of intensities of these bands should be about 1.5-2.5:1. Intact
mammalian poly (A)+ RNA appears as a smear sized from 0.1 to 4-7 (or more) kb with faint 28S and 18S
rRNA bands.
- In the case of RNA from non-mammalian sources (plants, insects, yeast, amphibians), the normal
mRNA smear on the non-denaturing agarose gel may not exceed 2-3 kb. Moreover, the overwhelming
majority of invertebrates have 28s rRNA with a so-called "hidden break" (Ishikawa, 1977). In some
organisms the interaction between the parts of 28s rRNA is rather weak, so the total RNA preparation
exhibits a single 18s-like rRNA band even on a non-denaturing gel. In other species the 28s rRNA is more
robust, so it is still visible as a second band.
Note: If your experimental RNA is shorter than expected and/or degraded according to electrophoresis
data, prepare fresh RNA after checking the quality of RNA purification reagents. If problems persist, you
may need to find another source of tissue/cells. In some cases, partially degraded RNA is only available
(e.g. tumor samples or hard treated tissues). This RNA can be used for cDNA preparation, however the
cDNA sample will contain reduced number of full-length molecules.
Total RNA from endothelial human cells.
- Commonly, genomic DNA contamination does not exceed the amount seen on the agarose/EtBr gel as a
weak band of
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
The analysis of global gene expression and transcription factor regulation, global approaches to alternative splicing and its regulation, long noncoding RNAs, gene expression models of signalling pathways, from gene expression to disease phenotypes, introduction to isoform sequencing, systematic and integrative analysis of gene expression to identify feature genes underlying human diseases.
The study of the complete set of RNAs (transcriptome) encoded by the genome of a specific cell or organism at a specific time or under a specific set of conditions is called Transcriptomics.
Transcriptomics aims:
I. To catalogue all species of transcripts, including mRNAs, noncoding RNAs and small RNAs.
II. To determine the transcriptional structure of genes, in terms of their start sites, 5′ and 3′ ends, splicing patterns and other post-transcriptional modifications.
III. To quantify the changing expression levels of each transcript during development and under different conditions.
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The simplified electron and muon model, Oscillating Spacetime: The Foundation...RitikBhardwaj56
Discover the Simplified Electron and Muon Model: A New Wave-Based Approach to Understanding Particles delves into a groundbreaking theory that presents electrons and muons as rotating soliton waves within oscillating spacetime. Geared towards students, researchers, and science buffs, this book breaks down complex ideas into simple explanations. It covers topics such as electron waves, temporal dynamics, and the implications of this model on particle physics. With clear illustrations and easy-to-follow explanations, readers will gain a new outlook on the universe's fundamental nature.
Read| The latest issue of The Challenger is here! We are thrilled to announce that our school paper has qualified for the NATIONAL SCHOOLS PRESS CONFERENCE (NSPC) 2024. Thank you for your unwavering support and trust. Dive into the stories that made us stand out!
Delivering Micro-Credentials in Technical and Vocational Education and TrainingAG2 Design
Explore how micro-credentials are transforming Technical and Vocational Education and Training (TVET) with this comprehensive slide deck. Discover what micro-credentials are, their importance in TVET, the advantages they offer, and the insights from industry experts. Additionally, learn about the top software applications available for creating and managing micro-credentials. This presentation also includes valuable resources and a discussion on the future of these specialised certifications.
For more detailed information on delivering micro-credentials in TVET, visit this https://tvettrainer.com/delivering-micro-credentials-in-tvet/
A review of the growth of the Israel Genealogy Research Association Database Collection for the last 12 months. Our collection is now passed the 3 million mark and still growing. See which archives have contributed the most. See the different types of records we have, and which years have had records added. You can also see what we have for the future.
Safalta Digital marketing institute in Noida, provide complete applications that encompass a huge range of virtual advertising and marketing additives, which includes search engine optimization, virtual communication advertising, pay-per-click on marketing, content material advertising, internet analytics, and greater. These university courses are designed for students who possess a comprehensive understanding of virtual marketing strategies and attributes.Safalta Digital Marketing Institute in Noida is a first choice for young individuals or students who are looking to start their careers in the field of digital advertising. The institute gives specialized courses designed and certification.
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This slide is special for master students (MIBS & MIFB) in UUM. Also useful for readers who are interested in the topic of contemporary Islamic banking.
How to Add Chatter in the odoo 17 ERP ModuleCeline George
In Odoo, the chatter is like a chat tool that helps you work together on records. You can leave notes and track things, making it easier to talk with your team and partners. Inside chatter, all communication history, activity, and changes will be displayed.
Natural birth techniques - Mrs.Akanksha Trivedi Rama University
Prabhakar singh ii sem-paper v-bioanalyzer
1. TEJASVI NAVADHITAMASTU
“Let our (the teacher and the taught) learning be radiant”
Let our efforts at learning be luminous and filled with joy, and
endowed with the force of purpose
Paper V: Instrumentation and Analytical Techniques
Bioanalyzer
2. Bioanalyzer
The Bioanalyzer is a chip-based capillary electrophoresis machine to analyse
RNA, DNA, and protein.
It is produced by Agilent and widely used, among other things, in RNA
quality control measurements before downstream experiments like
microarrays
Importance of Bio-analyzer
3.
4.
5.
6.
7.
8.
9.
10. RNA INTEGRITY NUMBER (RIN)
The RNA integrity number (RIN) is an algorithm for assigning integrity values
to RNA measurements. The integrity of RNA is a major concern for gene
expression studies and traditionally has been evaluated using the 28S to 18S rRNA ratio,
a method that has been shown to be inconsistent. This inconsistency arises because
subjective, human interpretation is necessary to compare the 28S and 18S gel images.
The RIN algorithm was devised to overcome this issue. The RIN algorithm is applied to
electrophoretic RNA measurements, obtained using capillary gel electrophoresis, and
based on a combination of different features that contribute information about the
RNA integrity to provide a more universal measure.
RIN has been demonstrated to be robust and reproducible in studies comparing it to
other RNA integrity calculation algorithms, cementing its position as a preferred
method of determining the quality of RNA to be analyzed
The RNA Integrity Database (RINdb) is a freely accessible repository holding hundreds
of user submitted total RNA traces. By searching the database scientists can now see
what is a "normal" profile for different tissue types as well as the effects of using
different RNA extraction methods and kits.
11.
12. Principle: RIN
The RNA integrity number (RIN) is a software tool designed to help scientists
estimate the integrity of total RNA samples.
The expert software automatically assigns an integrity number to an eukaryote
total RNA sample.
Using this tool, sample integrity is no longer determined by the ratio of the
ribosomal bands, but by the entire electrophoretic trace of the RNA sample.
This includes the presence or absence of degradation products. In this way,
interpretation of an electropherogram is facilitated, comparison of samples is
enabled and repeatability of experiments is ensured.
The assigned RIN is independent of sample concentration, instrument and
analyst therefore becoming a de facto standard for RNA integrity.
13. RIN Computation
RIN for a sample is computed using several characteristics of an RNA
electropherogram trace, with the first two being most significant. All the following
descriptions apply to mammalian RNA:
•Total RNA ratio
• Calculated by taking the ratio of the area under the 18S and 28S rRNA peaks
to the total area under the graph, a large number here is desired, indicating
much of the rRNA is still intact.
•Height of 28S peak
• Again, a large value is desired. 28S is used in RIN calculation as it is typically
degraded more quickly than 18S, and so measuring its peak height allows
for detection of the early stages of degradation.
•Fast area ratio
• The fast region is the area between the 18S and 5S peaks on an
electropherogram. Initially, as this value increases, it indicates degradation
of 18S and 28S rRNA to an intermediate size, though the ratio subsequently
decreases as RNA degrades further.
•Marker height
• A small number is desired here, indicating only small amounts of RNA have
been degraded and proceeded to the smallest lengths, indicated by the
short marker.
14. What is the meaning of the 28S/18S ribosomal ratios?
The 28S/18S ribosomal RNA ratio is frequently used to assess the
quality of total RNA purified from any given sample. The 28S and 18S
rRNAs are produced by the cleavage of a single RNA transcript.
Since they are produced from a single transcript, the ratio of the
number of 28S rRNA molecules to that of 18S molecules present in a
cell equals 1.
However, if you want to detect these rRNAs on an agarose gel, the
intensity of the bands produced by these molecules depends on the
number of nucleotides present in each molecule.
In humans, 28S rRNA has ~5070 nucleotides, and 18S has 1869
nucleotides, which gives a 28S/18S ratio of ~2.7. A high 28S/18S
ratio is an indication that the purified RNA is intact and hasn't
degraded. Usually, a 28S/18S ratio of >2 is taken to mean that the
purified total RNA is of high quality.
15.
16.
17. What the RIN can do:
1. Obtain a numerical assessment of the integrity of RNA.
2. Directly compare RNA samples, e.g. before and after archival, compare
integrity of same tissue across different labs.
3. Ensure repeatability of experiments, e.g. if RIN shows a given value and is
suitable for microarray experiments, then the RIN of the same value can
always be used for similar experiments given that the same
organism/tissue/extraction method is used.
What the RIN cannot do:
1. Tell a scientist ahead of time whether an experiment will work or not if no
prior validation was done (e.g. RIN of 5 might not work for microarray
experiments, but might work well for an appropriate RT-PCR experiment.
Also, a RIN that might be good for a 3' amplification might not work for a 5'
amplification).
18. A major criticism to RIN is when using with plants or in studies of eukaryotic-
prokaryotic cells interactions. The RIN algorithm is unable to differentiate
eukaryotic/ prokaryotic/ chloroplastic ribosomal RNA, creating serious quality
index underestimation in such situations
RNA integrity is critical for proper results in gene expression studies, such as
microarray analysis, Northern blots, or quantitative Real-Time PCR (qPCR).
qPCR and similar techniques are very expensive, taking a good deal of both time
and money, so continuing research being undertaken to decrease the cost while
maintaining qPCR's accuracy and reproducibility for gene expression and other
applications.
RIN assessment allows a scientist to evaluate an experiment’s trustworthiness
and reproducibility before incurring substantial costs in performing the gene
expression studies.
Applications
Drawback of RIN