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Questions and Answers
What is the primary site of transcription in prokaryotes?
What is the primary site of transcription in prokaryotes?
How many types of RNA polymerase are involved in eukaryotic transcription?
How many types of RNA polymerase are involved in eukaryotic transcription?
What modification does mRNA in eukaryotes undergo?
What modification does mRNA in eukaryotes undergo?
What is the first step in the total RNA isolation process?
What is the first step in the total RNA isolation process?
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What is Trizol primarily used for in RNA extraction?
What is Trizol primarily used for in RNA extraction?
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What is a major advantage of affinity purification in RNA extraction?
What is a major advantage of affinity purification in RNA extraction?
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How does DEPC function in the RNA extraction process?
How does DEPC function in the RNA extraction process?
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Which of the following is a limitation of organic extraction methods for RNA isolation?
Which of the following is a limitation of organic extraction methods for RNA isolation?
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What is the first modification added to a pre-mRNA transcript in eukaryotic cells?
What is the first modification added to a pre-mRNA transcript in eukaryotic cells?
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What process is responsible for removing introns from the pre-mRNA?
What process is responsible for removing introns from the pre-mRNA?
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Which enzyme is primarily involved in the capping of pre-mRNA?
Which enzyme is primarily involved in the capping of pre-mRNA?
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How does the 3' poly-A tail benefit pre-mRNA?
How does the 3' poly-A tail benefit pre-mRNA?
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Where does transcription occur in prokaryotic cells?
Where does transcription occur in prokaryotic cells?
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What is the role of the spliceosome in RNA processing?
What is the role of the spliceosome in RNA processing?
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Which of the following is NOT a post-transcriptional modification in eukaryotic cells?
Which of the following is NOT a post-transcriptional modification in eukaryotic cells?
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What do exons represent in the structure of RNA?
What do exons represent in the structure of RNA?
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What makes RNA less stable compared to DNA?
What makes RNA less stable compared to DNA?
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Which technique is used to estimate RNA quality after extraction?
Which technique is used to estimate RNA quality after extraction?
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What is a key step to suppress endogenous RNases during RNA isolation?
What is a key step to suppress endogenous RNases during RNA isolation?
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How do RNases affect RNA extraction procedures?
How do RNases affect RNA extraction procedures?
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What role does DEPC play in RNA isolation?
What role does DEPC play in RNA isolation?
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What is the purpose of adding Ethidium bromide to the electrophoresis gel?
What is the purpose of adding Ethidium bromide to the electrophoresis gel?
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At which wavelengths is RNA absorbance typically measured using a spectrophotometer?
At which wavelengths is RNA absorbance typically measured using a spectrophotometer?
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What characteristic of RNA is primarily responsible for its short half-life once extracted?
What characteristic of RNA is primarily responsible for its short half-life once extracted?
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Study Notes
RNA
- Ribonucleic acid (RNA) is a molecule present in most living organisms and viruses.
- RNA is made up of nucleotides, which consist of a ribose sugar, a nitrogenous base, and a phosphate group.
Post-Transcriptional Modifications
- Pre-mRNA is processed into messenger RNA (mRNA).
- Three main modifications are:
- 5' capping: 7-methylguanosine is added to the 5' end of the pre-mRNA.
- 3' poly-A tailing: A poly-A tail is added to the 3' end of the pre-mRNA.
- Splicing: Introns are removed from the pre-mRNA and exons are joined together.
Capping
- Capping is carried out by the addition of 7-methylguanosine.
- The addition of 7-methylguanosine is achieved by the removal of the terminal 5' phosphate, catalyzed by phosphatase enzymes.
- The enzyme adenosyltransferase facilitates the conversion of the 5' end into a diphosphate.
- Capping provides stability to the molecule, prevents degradation by ribonucleases, and aids mRNA recognition by the translation machinery.
Tailing
- The poly-A tail contributes to the stability of pre-mRNA.
- It signifies the end of transcription and aids in transporting the transcript to the cytoplasm.
Splicing
- Splicing enables the creation of new exon combinations, driving evolution.
- It regulates gene expression and protein content within the cell.
Transcription in Prokaryotes
- Transcription is a continuous process occurring in the cytoplasm.
- Only one type of RNA polymerase is involved in RNA synthesis.
- Post-transcriptional modifications are absent, and the mRNA lacks a 5' cap or a poly-A tail.
Transcription in Eukaryotes
- Transcription is a separate process, taking place in the nucleus.
- Three types of RNA polymerase enzymes are used during eukaryotic transcription.
- The produced mRNA has a 5' cap and a 3' poly-A tail.
RNA Isolation Importance
- RNA isolation methods are used for:
- Sequencing
- Size determination
- Expression level measurement
- Cloning
Methods of RNA Isolation
- Two main methods:
-
Organic Extraction:
- Employed for steady-state RNA isolation.
-
Affinity Purification:
- Used for nuclear run-off isolation.
-
Organic Extraction:
Steady-State RNA Isolation
-
Steps:
- Cell lysis: Using lysis buffer.
- De-proteinization: Using Trizol.
- Precipitation: Using isopropanol.
- Washing: Using 70% ethanol.
- Resuspension: Using RNase-free or DEPC-treated water.
Components Used in RNA Extraction
-
Trizol:
- Monophasic solution containing phenol, chloroform, and guanidinium isothiocyanate.
- Acidic phenol/ chloroform separates RNA into the aqueous supernatant.
- Guanidinium isothiocyanate denatures proteins and inactivates RNases.
-
DEPC:
- Reacts irreversibly with catalytic amino acids in the active site of RNase, inhibiting the enzyme.
- DEPC is classified as a suspected carcinogen and should be handled with caution.
Advantages of Affinity Purification
- Eliminates the need for organic solvents.
- Compatible with various sample types.
- DNase treatment eliminates contaminating DNA.
- High RNA purity and integrity.
Messenger RNA Isolation
- Isolation involves:
- Combining cytoplasmic RNAs with oligo(dT) matrix under hybridization conditions.
- The poly-A tail of mRNA binds to the oligo(dT) matrix.
- rRNA and tRNA are washed away.
- Purified mRNA is eluted from the oligo(dT) matrix using water or a low-salt buffer.
RNA Stability
- RNA is unstable and has a short half-life.
- Unstable due to:
- Single-stranded structure.
- Ribose sugar containing hydroxyl groups.
- Abundance of RNases.
Ribonuclease (RNase)
- Enzymes that catalyze RNA degradation.
- Present in blood, all tissues, bacteria, and fungi.
- Difficult to inactivate.
Successful RNA Isolation Requires
- Suppression of endogenous RNases.
- Prevention of contamination with exogenous RNases during extraction.
Suppression of Endogenous RNases
- Samples should be processed immediately or stored at -70°C or -80°C.
- Denaturing agents like urea, guanidinium hydrochloride, or guanidinium isothiocyanate are used to inactivate RNases.
Avoiding Contamination with Exogenous RNases During Extraction
- Use dedicated glassware, solutions, and equipment for RNA extraction.
- Treat water and laboratory utensils with DEPC, an RNase inhibitor.
- Autoclave glassware, solutions, and equipment if possible.
- Employ disposable gloves and RNAase-free disposable plastic materials.
Estimating RNA Quality
- Two techniques are used:
- Spectrophotometer.
- Gel electrophoresis.
Electrophoresis
- DNA and RNA possess negative charges.
- Following extraction, samples are subjected to horizontal denatured electrophoresis in an agarose gel.
- Ethidium bromide or SYBR green dyes are added to the gel, intercalating with the nucleotides of RNA or DNA.
- The dyes fluoresce under UV light.
Quantification
- Quantify extracted nucleic acids using a spectrophotometer:
- Measure RNA sample absorbance at 260nm and 280nm.
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