Derin Öğrenmeye Dayalı Çalışmalar

A presença de reação inespecífica em hibridização in situ é um problema constante para o uso desta técnica. A utilização de uma sonda senso controle teoricamente permite diferenciar o sinal plano de fundo, do sinal obtido com a sonda anti-senso hibridizada ao transcrito alvo. No entanto, esta não necessariamente é um controle efetivo. Genes podem ser transcritos nos dois sentidos, ou seja, é produzido transcrito complementar tanto a sonda anti-senso como senso (Coll

et al., 2010), resultando em sinal idêntico com ambas as sondas. Uma alternativa para a sonda

senso seria o uso de um mutante no qual o gene alvo não é transcrito, dessa forma, seria utilizado apenas a sonda anti-senso, tanto no espécime selvagem como no mutante. Uma vez que o mutante não apresenta o transcrito alvo, qualquer sinal obtido neste, seria sinal plano de fundo. Em Arabidopsis, estudos de hibridização in situ para o gene FIE (fertilization independent endosperm) foram realizados com apenas sonda anti-senso, sem controle senso, ou mutante fie, pois este era letal (Xu et al., 2016). Em comunicações não publicadas com pesquisadores que utilizaram ISH, foi constantemente relatado a aleatoriedade da obtenção de sinal, muitas vezes atribuída ao estado de desenvolvimento da amostra, assim como tamanho e sequência das sondas utilizada, ou mesmo a expressão do gene alvo em ambos os sentidos. Outra estratégia para diminuir a formação de sinal plano de fundo é o uso de uma concentração 10 vezes superior de sonda não marcada em relação a concentração de sonda-DIG, dessa forma o tecido é saturado com sondas não marcada, dificultando a interação inespecífica da sonda-DIG (Bagasra, 2007; Landegent et al., 1987). Uma vez que as sondas preferencialmente interagem com o mRNA alvo em relação a interações inespecíficas, a saturação do tecido com sondas não marcadas diminuiria a formação de sinal plano de fundo. Outra estratégia seria a combinação dos protocolos de PCR e hibridização in situ. Possivelmente, realizar a RT-PC in situ utilizando nucleotídeos não marcados e em seguida, essas secções contendo os produtos de PCR seriam submetidas a ISH com a sonda anti-senso apenas, na esperança de que o acúmulo da sequência alvo poderia favorecer a obtenção de sinal específico.

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