mol bio 2 lecture 2 questions

What is the purpose of transcription assays in studying gene expression?

To analyze RNA synthesis

Which method is commonly used for detecting proteins?

Western blot

What does immunoprecipitation (IP) help study in protein interactions?

Protein interactions

What is the purpose of chromatin immunoprecipitation (ChIP)?

To study protein-DNA interactions

Which assay is used to analyze the binding of proteins to nucleic acids?

Gel shift assay (EMSA)

What is the primary purpose of isolating RNA in cell culture?

To detect specific RNAs in intact cells or tissues

What does an RNA sample consist of?

A mixture of all the RNAs in the cell

Which institution is mentioned as a source for detecting specific RNAs in RNA samples?

Mayo Clinic

What is RNA hybridization?

The process of RNA molecules binding to complementary sequences

What are some methods for detecting RNA levels?

FISH

Which technique commonly utilizes RNA hybridization?

Northern blotting

What is the significance of complementary sequences in RNA hybridization?

They allow for the formation of stable double-stranded RNA

What is the purpose of the Northern blot technique?

To transfer RNA onto a membrane

What is the first step in the Northern blot process?

Perform agarose gel electrophoresis

What happens to RNA bands during the Northern blot process?

They are transferred to a membrane

What technique is used to visualize the RNA bands after they are transferred to the membrane?

Autoradiography

What molecule's effect on the expression of the gene DICER was investigated in the study?

Homocysteine

What method was used to quantify the expression levels of DICER in the study?

Densitometry

What was used as a loading control in the RT PCR performed in the study?

GAPDH

How were the results of the experiments presented in the study?

Averaged with standard deviations

What type of cells were treated with homocysteine in the study?

Cardiomyocytes

What is the purpose of transcription assays in studying gene expression?

To measure RNA levels

Which method is commonly used for detecting proteins?

Western blot

What is the function of epitope tagging in protein studies?

To facilitate protein detection

Which assay is used to study protein interactions?

Immunoprecipitation (IP)

What does the chromatin immunoprecipitation (ChIP) assay help to analyze?

DNA-protein interactions

What does the steady state RNA level indicate?

All of the above

Which method is NOT mentioned as a way to detect RNA levels?

Western blot

What is the relationship between steady state RNA levels and transcription rates?

They do not necessarily reflect each other

What does NRO stand for in the context of gene transcription analysis?

Nuclear Run On

What type of RNA does NRO assays focus on?

Nascent RNA

What is the relationship between RNA polymerase (RNAP) molecules and nascent RNA levels in NRO analysis?

More RNAPs lead to higher nascent RNA levels

Why is nascent RNA analysis preferred over total steady state RNA levels in NRO assays?

It provides a better picture of gene activity

What is the first step in the Nuclear Run On (NRO) procedure?

Nuclei are isolated from cells

What happens to RNAPs when NTPs are depleted during the NRO procedure?

They are prevented from binding to DNA

What role do labeled NTPs play in the NRO procedure?

They allow halted RNAPs to continue transcription

What is the effect of adding sarkosyl during the NRO procedure?

It blocks unengaged RNAP from binding to DNA

Which of the following nucleotides are referred to as NTPs in the context of transcription?

ATP, CTP, GTP, UTP

What does NRO stand for in the context of RNA isolation?

Nuclear Run On

What is the purpose of identifying and quantifying labeled NRO transcripts?

To identify active genes and their activity levels

Which techniques can be used to identify and quantify labeled nascent RNAs?

RT PCR and RNA seq

What is the combination of NRO and RNA seq referred to as?

GRO seq

What is a special feature of the label used in NRO RNA isolation?

It allows for purification of labeled RNAs from other RNAs

What is the purpose of the Nuclear Run On (NRO) technique?

To label only nascent RNAs

What happens to the polymerase during the NRO experiment when NTPs are not available?

It pauses transcription

Which gene was confirmed to be transcriptionally active in the NRO experiment?

Gene Y

What method was used to analyze the isolated labeled RNAs in the NRO experiment?

RT PCR or RNA seq

What is the purpose of transcription assays in studying gene expression?

To analyze RNA synthesis

Which method is commonly used for labeling nucleic acids?

In vitro transcription

What technique is used to detect proteins in a sample?

Western blot

What is the purpose of immunoprecipitation (IP)?

To isolate specific proteins

Which assay is used to study protein-DNA interactions?

Chromatin immunoprecipitation (ChIP)

What is the primary purpose of in vitro transcriptional analysis?

To study gene expression in a cell-free system

What advantage does in vitro transcription provide compared to in vivo methods?

It enables direct study of transcription without cellular complexity

What does in vitro transcriptional analysis help to quantify?

Gene expression

What is the primary purpose of in vitro transcription analysis?

To study genes/promoters and transcription factors

Which components are typically included in the in vitro transcription reaction?

Template DNA, NTPs, RNA Polymerase, Transcription Factors

How is the amount of RNA transcript produced measured in in vitro transcription analysis?

By the gene/promoter activity reflected in the RNA transcript

What method is commonly used to detect the RNA products of in vitro transcription?

Electrophoresis followed by autoradiography

In PAGE, which nucleic acids travel farther?

Smaller nucleic acids

What is the role of the transcription factor NTF in the study?

To determine its role in transcription

What does the term 'novel' refer to in the context of the transcription factor NTF?

Something that has not been reported or studied before

What components are included in the transcription buffer used for in vitro transcription?

Salts, buffer, and necessary chemicals

What is the purpose of the human nuclear extract in the transcription reactions?

To facilitate transcription

What does the term 'buffer' technically refer to in molecular biology?

An aqueous solution of a weak acid and its conjugate base

What is the role of NTF in transcription according to the study?

Activator of RNAP II transcription

What method was used to analyze the products of the transcription reactions?

PAGE electrophoresis and autoradiography

What was the experimental approach taken to study the effects of NTF?

In vitro transcription reactions with varying amounts of NTF

How long were the transcription reactions incubated during the study?

Several minutes

What is the primary purpose of in vitro transcription analysis?

To study genes/promoters and transcription factors

Which of the following components is NOT typically included in an in vitro transcription reaction?

DNA Ligase

Which nucleotides are used during the transcription process with the G less cassette?

ATP, CTP, UTP

How is the amount of RNA transcript produced in an in vitro transcription analysis measured?

By the reflection of gene/promoter activity

What method is commonly used to detect the products of in vitro transcription?

Electrophoresis

In PAGE, which nucleic acids travel farther?

Smaller nucleic acids

What is a common type of in vitro transcription template mentioned in the text?

Run Off Transcription

What is Run Off Transcription Analysis?

A type of in vitro transcriptional analysis

What does RNAP bind to during Run Off Transcription?

Promoter

What is the transcriptional start site (TSS)?

The first nucleotide/base read and copied into RNA

What generates a restriction fragment of DNA?

Restriction endonucleases

What does NTP stand for in the context of transcription analysis?

Nucleotide Triphosphate

What is the length of the RNA fragment produced in the example provided?

327 nt

What does TSS stand for in the context of transcription analysis?

Transcription Start Site

What is the distance from the TSS to the distal end of the restriction fragment in the example?

327 bp

What is the role of transcription factors (TFs) in eukaryotic systems?

They establish the correct transcription start site (TSS)

What method can be used to demonstrate the necessity of certain transcription factors?

In vitro transcription

What is the primary purpose of the G less cassette in transcription analysis?

To analyze transcription in vitro

What happens when RNAP encounters the first G outside of the G less cassette?

Transcription stops

What is a significant advantage of using the G less cassette compared to traditional methods?

It generates a transcript of defined length

What other types of cassettes could be used similarly to the G less cassette?

A less cassette, C less, or T less cassette

What is the purpose of transcription assays in studying gene expression?

To analyze RNA synthesis

Which method is commonly used for detecting proteins?

Western blot

What is the function of epitope tagging in protein studies?

To facilitate protein purification

What does the chromatin immunoprecipitation (ChIP) technique study?

Protein-DNA interactions

Which assay is used to study protein interactions through co-immunoprecipitation (Co-IP)?

Immunoprecipitation

What is the purpose of labeling nucleic acids in molecular biology?

To allow detection of nucleic acids

Which of the following techniques uses labeled nucleic acids for detection?

Northern blot

In an NRO experiment, what can labeled nascent RNAs be purified from?

Total RNA samples

What is one application of labeled nucleic acids besides detection?

Purifying specific nucleic acids

What is a characteristic of radioactive isotopes used in labeling nucleic acids?

They can be incorporated into nucleic acids in vitro